HELMINTH PARASITE ANTIGEN WITH AMINOPEPTIDASE-LIKE ACTIVITY

Abstract: A protective helminth parasite antigen which is characterised by processing aminopeptidase-like activity and which, in native form, is an integral membrane protein associated with the gut microvilli of a non-obligate blood feeding helminth parasite, or a functionally-equivalent variant, antigenic fragment or precursor thereof methods for its perparation, vaccine compositions containing it and its use in stimulating an immune response against hellminth parasites in a human or non-human animal.

Description: HELMINTH PARASITE ANTIGEN WITH AMINOPEPTIDASE-LIKE ACTIVITY . Vaccines This invention relates to novel helminth parasite antigens and their use in the control of disease caused by helminth parasites, particularly parasitic nematodes of the gastro-intestinal tract of mammals. Helminth parasites, particularly nematodes, infect or infest a wide range of animals, including man, and are a widespread and significant source of disease and ill-thrift, not only in animals, but also in man. Such parasites thus represent a considerable worldwide drain on economic resources. This is particularly true in animal husbandry, where parasite infections of grazing animals, such as sheep and cattle, are often difficult and expensive to control and may result in significant economic losses. Particular mention may be made in this regard of the non-blood feeding nematodes Ostertaσia ostertaαi and Qster agia (Telaάorsagia) circumcincta ' (O. circumcincta has recently been reclassified as T.circumcincta. although the new name is not yet in wide usage) . Other parasitic helminths of economic importance include the various species of the following helminth families:- TrichQstrQngylus, Ne atoάirus, Dictyocauius. COPPeria, TrichuriS, Oesophaσostomum. Bunostomum and Metastronσylus. In addition to domestic livestock, pets and humans may also be infected, not infrequently with fatal results. At present, control of helminth parasites of grazing livestock relies primarily on the use of anthelmintic drugs, combined with pasture management. Such techniques have not proved entirely satisfactory however, due to their expense and inconvenience and to a rapid increase in drug resistance. Anthelmintic drugs need to be administered frequently and appropriate pasture management is often not possible on sone farms and even where it is, it can place constraints on the best use of available grazing. To overcome these problems, attempts have been made to achieve immunological means of control. Although there has been some success in identifying certain protective antigens as potential vaccine candidates, most notably in Haemonchus. this approach has proved difficult and, other than for the cattle lungworm Dictyocaulus viviparus. has yet to come to commercial fruition. The most success to date has been achieved with the protein doublet H110D, an integral membrane protein isolated from the gut of H.contortus and described by Munn in O88/00835. H110D now represents the most promising vaccine candidate to date. Munn has also described and proposed as a vaccine, contortin, a helical polymeric extracellular protein associated with the luminal surface of H.contortus intestinal cells (Munn et al.. Parasitology 2 : 385-397, 1987) . A third Haemonchus gut membrane protein with protective antigenic properties has also been discovered and termed H45 (Munn and Smith, WO90/11086) . Whilst proteins such as H110D and H45 can be used as the basis for a vaccine against Haemonchus , there is nonetheless a continuing need for new and improved helminth parasite vaccines and in particular for a vaccine which may be used across a broad range of helminth genera. Most studies to date have concentrated on blood feeding nematodes such as Haemonchus and there is especially a need for vaccines against non-blood feeding helminths such as TrichPStrongylUS, Cooperia. Dictyocaulus. and particularly Ostertagia. The present invention accordingly seeks to provide novel antigens for use as helminth parasite vaccines and in particular as protective im unogens in the control of diseases caused by non-blood feeding helminth parasites. More specifically, the present invention is based on the finding that the gut of non-blood feeding nematodes such as Ost-.ertaαia and Cooperia contains an integral membrane protein having aminopeptidase activity which is believed to be an important protective antigen capable of conferring protective immunity against helminth parasites in animals. Such proteins, when liberated from the membranes in which they are integral, for example by the use of detergents, are novel and of use in the manufacture of vaccines against helminth infections. According to one aspect, the present invention thus provides a protective helminth parasite antigen which is characterised by possessing aminopeptidase-like activity and which, in native form, is an integral membrane protein associated with the gut microvilli of a non- obligate blood feeding helminth parasite, or a functionally-equivalent variant, or antigenic fragment or precursor thereof. A further aspect of the invention provides such protective antigens, and functionally-equivalent variants, antigenic fragments or precursors thereof, for use in stimulating an immune response against helminth parasites in a human or non-human, preferably mammalian, especially preferably ruminant, animal. A precursor for the antigen in question may be a larger protein which is processed, eg. by proteolysis, to yield the antigen per se. Such precursors may take the form of zymogens ie. inactive precursors of enzymes, activated by proteolytic cleavage, for example analogous to the pepsin/pepsinogen system or the well known zymogens involved in the blood clotting cascade. Non-obligate blood feeding parasites are defined for present purposes as those which imbibe host blood rarely and seemingly incidentally (in contrast to obligate blood feeders such as Haemonchus , which feed almost exclusively on host blood) and include both plug feeders and browsers. Plug-feeding worms have enlarged buccal capsules which they use to envelope a plug of host tissue e.g. the intestinal mucosa in the case of Chabertia ovina) from which they obtain nutrient. Browsing nematodes do not have mouthparts specialised for attachment. The composition of their diet is not known but is thought to consist either of host tissue, extracellular tissue fluid, mucus or, in the case of gastro-intestinal species, host digesta. Some gastrointestinal species, particularly those which live in close association with the mucosa (e.g. Osteragia circumcincta in the abomasal glands and certain Trichostronσylus species (e.g. other than T.axei) which tunnel under the intestinal epithelium may feed on a mixture of these. Economically important helminth parasites which are not obligate blood feeders include the following genera and their subspecies:- Ostertagia (which, for the avoidance of doubt, as used herein includes Teladprsagia) , Trichpstrpngylus spp. (e.g. T.colubriformis. T.vitrinUS and T.axei) , Cooperia spp. , Nβmat dir s, Chabertia and Qespphagpstpmum■ The novel antigens of the invention are not recognised by sera from naturally immune animals. In other words, they are not normally, in native form, accessible to the immune system of the infected host and are thus "hidden", "concealed" or "cryptic" antigens. The term "protective antigens" or "protective antigenic activity" as used herein defines those antigens and their fragments or precursors, capable of generating a host-protective, ie. immunogenic, immune response, that is a response by the host which leads to generation of immune effector molecules, antibodies or cells which damage, inhibit or kill the parasite and thereby "protect" the host from clinical or sub-clinical disease and loss of productivity. Such a protective immune response may commonly be manifested by the generation of antibodies which are able to inhibit the metabolic function of the parasite, leading to stunting, lack of egg production and/or death. As mentioned above, included within the scope of the invention are functionally-equivalent variants of the novel antigens and their fragments and precursors. "Functionally-equivalent" is used herein to define proteins related to or derived from the native protein, where the amino acid sequence has been modified by single or multiple amino acid substitution, addition and/or deletion and also sequences where the amino acids have been chemically modified, including by deglycosylation or glycosyla ion, but which nonetheless retain protective antigenic activity eg. are capable of raising host protective antibodies and/or functional immunity against the parasites. Within the meaning of "addition" variants are included amino and/or carboxyl terminal fusion proteins or polypeptides, comprising an additional protein or polypeptide fused to the aminopeptidase antigen sequence. Such functionally- equivalent variants mentioned above include natural biological variations (eg. allelic variants or geographical variations within a species) and derivatives prepared using known techniques. For example, functionally-equivalent proteins may be prepared either by chemical peptide synthesis or in recombinant form using the known techniques of site- directed mutagenesis, random mutagenesis, or enzymatic cleavage and/or ligation of nucleic acids. Functionally-equivalent variants according to the invention also include analogues in different parasite genera or species. Aminopeptidase enzymes are believed to function as part of the nutrient assimilation process of helminths and blockage of enzymic activity may contribute to the protective effect of the antibodies induced in response to immunisation with the antigens of the invention. Aminopeptidase-like activity may be assayed using a range of substrates, for example alanine, leucine and α- glutamyl-p-nitroanilide. Antigens of the invention have been shown to possess a range of aminopeptidase activities, most notably aminopeptidase M(ApM)-like and aminopeptidase A (ApA) -like activity, which may be demonstrated using e.g. alanine, leucine or methionine p-nitroanilide and α-glutamic acid p-nitroanilide respectively, for example in helminth extracts or on substrate-gels following polyacrylamide-gel electophoresis (PAGE) using corresponding β-methoxy naphthylamide substrates. In addition, inhibitor studies have shown the antigens to be metalloproteinases. The aminopeptidase antigens of the invention are integral membrane proteins, as may be shown by detergent solubilization studies. For example, the antigens are insoluble in aqueous buffers, such as phosphate-buffered saline (PBS) and detergents such as polyoxyethylene- sorbitan-monolaurate (Tween) which solύbilise membrane- associated proteins, but may be solubilised by non-ionic detergents, e.g. TRITON X-100, or dodecylpolyethylene glycolether (THESIT, from Desitin Werk, Hamburg, Germany) which solubilise integral membrane proteins. Histochemical studies have shown the antigens of the invention to be associated with the intestinal microvilli of the parasites. Thus, for example in the non-obligate blood feeding helminths e.g. Ostertaσia and Cooperia. staining of frozen sections reveals the presence of aminopeptidase activity in the area of the intestinal microvilli. Lectin binding studies have further shown that the antigens of the invention bind to concanavalin A (ConA) , indicating the presence of glycosylation, particularly mannose and/or fucose residues. Preferred antigens according to the invention are those isolatable from Ostertaσia. In particular, in both 0.circumcincta and Q.PStertagi aminopeptidase activity (including ApA- and ApM-like) has been shown to be associated with a band of material which may be separated by electrophoresis on polyacrylamide gel under denaturing conditions, and which has an apparent molecular weight (Mr) of about 124,000-126,000 (6-16% SDS-PAGE) . Such proteins and their analogues in other non-blood feeders represent particularly preferred embodiments of the invention. Antigens according to the invention may be obtained by extraction of non-obligate blood feeding helminth parasites using appropriate detergents. A further aspect of the invention thus provides a method for preparing the above-mentioned antigens of the invention which comprises the step of subjecting a crude extract of a non-obligate blood feeding helminth parasite to detergent extraction, eg. using Tween 20 or a like detergent to remove membrane-associated proteins followed by Thesit or a like detergent, and recovering the solubilised integral membrane proteins. The detergent extract may then be subjected to further purification using conventional procedures eg. centrifugation, selective precipitation, electrophoresis, chromatography and the like. Fractions containing the antigen of the invention may be identified by assaying for aminopeptidase activity using known techniques. The antigen may be released from the membrane in a truncated form, which is soluble in water without addition of detergent, by treatment with a proteolytic enzyme such as trypsin. Antigens obtainable by such methods form a further aspect of the invention. The crude extract of the helminth parasite may be prepared using conventional biochemical and surgical techniques eg. by homogenisation of the whole or a portion of the parasite. Thus, for example the parasites may be subjected to homogenisation in a suitable buffer or medium such as phosphate buffered saline (PBS), and the insoluble material (ie. the pellet) may be recovered by centrifugation, whereby to form the required crude extract. Thus, a suitable purification protocol for the antigens of the invention might comprise (i) homogenising adult parasite worms in PBS, recovering the PBS-insoluble material (eg. by centrifugation) , optionally repeating the PBS-washing step one or more times, (ii) extracting the PBS-insoluble material (ie. the pellet) with aqueous Tween 20 (eg. in PBS) , (iii) recovering the Tween-insoluble fraction e.g. by centrifugation, (iv) extracting the Tween-insoluble material with aqueous Thesit (e.g. in PBS) , (v) recovering the solubilised fraction containing the Thesit-soluble proteins, (ie. the supernatent also known as ThHS) followed by (vi) ion exchange chromatography eg. on a Mono Q column, and/or gel filtration eg. using Superose. A ConA affinity chromatography step may optionally also be included e.g. before ion exchange. An alternative preparation process may take advantage of the fact that the target antigens have particular selective binding activities by using an affinity chromatography system in which specific ligands are immobilised on a solid phase matrix. Thus the invention also provides a process for preparing the above-mentioned antigens of the invention, said process comprising preparing an extract of said parasite containing at least one protective antigen as defined above, purifying said antigen therefrom by binding said antigen to an immobilized phase including a specific binding partner for the antigen and subsequently eluting said antigen from said immobilized phase. Suitable specific binding partners include substrate analogues, for example the inhibitor bes atin, and oligosaccharide specific leetins e.g. ConA. The invention also provides use of a helminth parasite antigen as hereinbefore defined, and fragments, precursors and functionally-equivalent variants thereof, for the preparation of a vaccine composition for use in stimulating an immune response against helminth parasites in a human or non-human, animal. Further provided according to the invention is a vaccine composition for stimulating an immune response against helminth parasites in a human or non-human animal comprising one or more antigens, antigenic fragments, precursors or functionally-equivalent variants thereof, as defined above, together with a pharmaceutically acceptable carrier or diluent, and a method of stimulating an immune response against helminth parasites in a human or non-human animal, comprising administering to said animal a vaccine composition as defined above. The animal preferably is mammalian and more preferably a ruminant. As mentioned above, antigens according to the invention may be obtained from a range of non-obligate blood feeding helminth parasite genera. Preferably, however the helminths will be nematodes, especially preferably gastro-intestinal nematodes including for example Ostertaσia and CPPPeri ■ As mentioned above, antigens from Ostertaσia are especially preferred. Such antigens may be used to prepare vaccines against a range of helminth parasites including any of those mentioned above. Preferred are those antigens, so called "broad spectrum" antigens, which are capable of stimulating host protective immune responses against, in addition to the parasite from which they were isolated, a broad range of other parasites. As mentioned above, one of the ways in which the antigens of the invention may exert their host protective effects is by raising inhibitory antibodies which inhibit the growth, maintenance and/or development of the parasite. Such antibodies and their antigen- binding fragments (eg. F(ab) 2 , Fab and Fv fragments ie. fragments of the "variable" region of the antibody, which comprises the antigen binding site) which may be mono- or polyclonal, form a further aspect of the invention, as do vaccine compositions containing them and their use in the preparation of vaccine compositions for use in passively immunising hosts against parasites. Such inhibitory antibodies may be raised by use of idiotypic antibodies. Anti-idiotypic antibodies may be used as immunogens in vaccines. In addition to the extraction and isolation techniques mentioned above, the antigens may be prepared by recombinant DNA technology using standard techniques, such as those described for example by Sambrook et al. , 1989, (Molecular Cloning, a laboratory manual 2nd Edition, Cold Spring Harbor Press) . Nucleic acid molecules comprising a nucleotide sequence encoding the antigens of the invention thus form further aspects of the invention. Nucleic acid molecules according to the invention may be single or double stranded DNA, cDNA or RNA, preferably DNA, and include degenerate, substantially homologous and hybridising sequences which are capable of coding for the antigen or antigen fragment or precursor concerned. By "substantially homologous" is meant sequences displaying at least 60%, preferably at least 70% or 80% sequence homology. Hybridising sequences included within the scope of the invention are those binding under non-stringent conditions (6 x SSC/50% formamide at room temperature) and washed under conditions of low stringency (2 x SSC, room temperature, more preferably 2 x SCC, 42°C) or conditions of higher stringency eg. 2 x SSC, 65°C (where SSC = 0.15M NaCl, 0.015M sodium citrate, pH 7.2), as well as those which, but for the degeneracy of the code, would hybridise under the above-mentioned conditions. Derivatives of nucleotide sequences capable of encoding antigenically active antigens or antigen variants according to the invention may be obtained by using conventional methods well known in the art. Antigens according to the invention may be prepared in recombinant form by expression in a host cell containing a recombinant DNA molecule which comprises a nucleotide sequence as broadly defined above, operatively linked to an expression control sequence, or a recombinant DNA cloning vehicle or vector containing such a recombinant DNA molecule. Synthetic polypeptides expressed in this manner form a further aspect of this invention (the term "polypeptide" is used herein to include both full-length protein and shorter length peptide sequences) . The antigen so expressed may be a fusion polypeptide comprising all or a portion of an antigen according to the invention and an additional polypeptide coded for by the DNA of the recombinant molecule fused thereto. This may for example be β-galactosidase, glutathione-S-transferase, hepatitis core antigen or any of the other polypeptides commonly employed in fusion proteins in the art. Other aspects of the invention thus include cloning and expression vectors containing the DNA coding for an antigen of the invention and methods for preparing recombinant nucleic acid molecules according to the invention, comprising inserting nucleotide sequences encoding the antigen into vector nucleic acid, eg. vector DNA. Such expression vectors include appropriate control sequences such as for example translational (eg. start and stop codons, ribosomal binding sites) and transcriptional control elements (eg. promoter-operator regions, termination stop sequences) linked in matching reading frame with the nucleic acid molecules of the invention. Vectors according to the invention may include plasmids and viruses (including both bacteriophage and eukaryotic viruses) according to techniques well known and documented in the art, and may be expressed in a variety of different expression systems, also well known and documented in the art. Suitable viral vectors include baculovirus and also adenovirus and vaccinia viruses. Many other viral vectors are described in the art. A variety of techniques are known and may be used to introduce such vectors into prokaryotic or eukaryotic cells for expression, or into germ line or somatic cells to form transgenic animals. Suitable transformation or transfection techniques are well described in the literature. The invention also includes transformed or transfected prokaryotic or eukaryotic host cells, or transgenic organisms containing a nucleic acid molecule according to the invention as defined above. Such host cells may for example include prokaryotic cells such as E.coli. eukaryotic cells such as yeasts or the baculovirus-insect cell system, transformed mammalian cells and transgenic animals and plants. Particular mention may be made of transgenic nematodes (see for example Fire, 1986, EMBO J., ≤.. 2673 for a discussion of a transgenic system for the nematode Caenorhabdii-.-is.. A further aspect of the invention provides a method for preparing an antigen of the invention as hereinbefore defined, which comprises culturing a host cell containing a nucleic acid molecule encoding all or a portion of said antigen, under conditions whereby said antigen is expressed and recovering said antigen thus produced. The antigens of the invention and functionally equivalent antigen variants may also be prepared by chemical means, such as the well known Merrifield solid phase synthesis procedure. Water soluble derivatives of the novel antigens discussed above form a further aspect of the invention. Such soluble forms may be obtained by proteolytic digestion, for example using the enzyme trypsin. Generally speaking enzymic digestion of the antigens yields two fractions, a detergent soluble fraction (which remains with the membrane) and a water-soluble fraction. A vaccine composition may be prepared according to the invention by methods well known in the art of vaccine manufacture. Traditional vaccine formulations may comprise one or more antigens or antibodies according to the invention together, where appropriate, with one or more suitable adjuvants eg. aluminium hydroxide, saponin, quil A, or more purified forms thereof, muramyl dipeptide, mineral or vegetable oils, Novasomes or non-ionic block co-polymers or DEAE dextran, in the presence of one or more pharmaceutically acceptable carriers or diluents. Suitable carriers include liquid media such as saline solution appropriate for use as vehicles to introduce the peptides or polypeptides into an animal or patient. Additional components such as preservatives may be included. An alternative vaccine formulation may comprise a virus or host cell eg. a microorganism (eg. vaccinia virus, adenovirus, bacteria such as the Bacillus Calmette-Guerin strain of Mycobacterium bovis (BCG) or Salmonella spp) which may be live, killed or attenuated, having inserted therein a nucleic acid molecule (eg. a DNA molecule) according to this invention for stimulation of an immune response directed against polypeptides encoded by the inserted nucleic acid molecule. Administration of the vaccine composition may take place by any of the conventional routes, eg. orally or parenterally such as by intramuscular injection, optionally at intervals eg. two injections at a 7-35 day interval. The antigens may be used according to the invention in combination with other protective antigens obtained from the same or different parasite species. Such a combined vaccine composition may contain smaller amounts of the various antigens than an individual vaccine preparation, containing just the antigen in question. Combined vaccines are beneficial where there is a likelihood that "adaptive selection" of the parasite may occur when a single antigen vaccine is used. Animals which may benefit from the present invention may be any human or non-human animal, but companion animals, particularly dogs and cats and domestic animals, especially ruminants are preferred. Particular mention may be made of sheep, cattle, pigs, deer and goats. Treatment of membrane preparations obtained from 0. ostertaσi with trypsin released aminopeptidase M activity in a water-soluble form which bound to ConA and after gel filtration yielded a fraction with high activity and showing a Coomassie blue staining band on SDS-PAGE around 116,000. The N-terminal sequence of a band of similar size obtained by SDS-PAGE of a breakdown product of 012 was Ala Glu Asp Leu Arg Leu Pro Thr Asn He Arg Pro Leu He Tyr Asp Leu Thr (AEDLRLPTNIRPLIYDLT, Sequence I.D. No. 1) . This shows 67% identity and 89% similarity to the sequence of the corresponding region of microsomal aminopeptidases HllOD-1, -2 and -3 from Haemonchus contortus deduced from their DNA sequences and 67% identity to the observed N-terminal amino acid sequence of a water soluble fragment released from HllOD by trypsin cleavage. The amino acids Leu, Pro, Pro and Tyr at positions 6, 7, 12 and 15 respectively of this peptide are conserved in human, pig, rat and mouse microsomal aminopeptidases. The full sequence of the 012 microsomal aminopeptidase was obtained by sequencing cloned PCR products generated from Ostertaσia ostertaσi first strand cDNA and primers based on a) the sequence GAMENWGL (Sequence I.D. No. 2) common to the HllOD microsomal aminopeptidases, b) at the 5' end, the helminth spliced leader sequence SL1 and c) a vector sequence at the 3' end. Antibodies to 012 do not form in the course of infection but the protein is shown to be highly immunogenic in sheep and cattle when injected in purified form. Antibodies raised against 012 from 0. ostertaσi cross-react with periodate-treated 012 from 0. circumcincta as do antibodies raised in sheep to CamQHHOD. This invention will now be described in more detail with particular reference to the protein 012 from Ostertaσia ostertaσi. However, by a variety of techniques such as histochemistry and Western blotting, 012 equivalents have been demonstrated in the parasites Ostertaσia πircumcincta and Cooperia oncophora. It is believed that the 012 protein is a multigene complex and that in addition, the nucleotide sequences encoding it, may exhibit sequence variations between different strains and different life cycle stages of the helminth. Moreover there may exist multiple enzyme forms (isoenzymes) which may be differentially expressed at different stages, or in different strains. In this study DNA sequences, and thus the predicted amino acid sequences, have been determined from cDNA clones and PCR products obtained from mRNA corresponding to the 012 gene by recombinant DNA technology from 12-days old (early fifth stage) Ostertaσia ostertaσi. Sequencing of PCR products has enabled us to identify closely related 012 sequences which are here designated 012-1 (Sequence I.D. No. 19), 012-2 (Sequence I.D. 20), 012-3 (Sequence I.D. No. 21), 012-4 (Sequence I.D. No. 22) and 012-5 (Sequence I.D. No. 23) . 012-1 and 012-2 are 5' sequences of about lkb terminating at their 3 ' ends in the primer based on the conserved sequence GAMENWGL (Sequence I.D. No. 2); 012-3, 012-4 and 012-5 are 3' sequences of about 2.5kb originating at their 5' ends with this primer. The relationships of 012-1 and -2 with 012-3, -4 and -5 may be determined by PCRing between primers towards the 3' ends of 012-1 and -2 and the 5' ends of 012-3, -4 and -5 to include the common GAMENWGL (Sequence I.D. No. 2) sequence. Differences and variations in the sequences obtained from the PCR products have been observed, as can be seen in particular from Figures 19 and 20 (Sequence I.D. Nos. 19-23) and as summarised below. Homologies of the deduced amino acid sequences (Sequence I.D. Nos. 24-28) obtained by translation of the nucleotide sequences shown in Figures 19 and 20 (Sequence I.D. Nos. 19-23). The differences can be attributed to different mRNAs multigene family. Comparison of the sequences of various of the clones mentioned above, against computer databases of known sequences, reveals substantial homology with the family of microsomal aminopeptidase enzymes (EC. 3.4.11.-). Enzymological activity and inhibitor studies performed with the 012 protein and subfractions thereof confirm that the protein is in fact microsomal aminopeptidase (α-amino acyl peptide hydrolase (microsomal) ) . Using the enzyme trypsin, it was found that 012 may be partially cleaved, forming two fractions, a detergent-soluble fraction (which remained with the membrane) and a water-soluble fraction (which is designated 012S) ; it was found that aminopeptidase M- like activity is associated with the water-soluble 012S fraction. An eighteen amino acid N-terminal sequence (designated Pep A, Sequence I.D. No. 1) from a proteolytic fragment of 012 has 67% identity and 89% similarity to the corresponding fragments of HllOD-1, -2 and -3 deduced from their DNA sequences. The sequences are PepA: AEDLRLPTNIRPLIYDLT Hll-1: AEELRLPTTIKPLTYDLV PepA: AEDLRLPTNIRPLIYDLT Hll-2: AEELLLPTNIKPVSYDLN PepA: AEDLRLPTNIRPLIYDLT Hll-3: AAELLLPSNIKPLSYDLT Over this region the three deduced Hll sequences are 72-77% identical (13 or 14 amino acids) and 83-100% similar.) Nine amino acids are conserved in all four sequences: A12L3LP45I6P7XYDLX For the other residues, 1 is either E or A, 2 is D in PepA and E in Hll, 3 is L or R, 5 is N or T, 6 is R in PepA and K in Hll, and 7 is L or V ( LP P Y are conserved in human, pig, rat and mouse microsomal aminopeptidases.) Comparing the amino acid sequences of PepA (Sequence I.D. No. 1) and HllOD (Hll) as determined we have: PepA AEDLRLPTNIRPLIYDLT (N-terminus of a breakdown product . ) Hll AEELLLPTNIKPVSYDLK (N-terminuε of a trypsin fragment . ) this has 67% identity and 83% similarity. PCR from 0. ostertaαi first strand cDNA using an oligonucleotide corresponding to the HllOD conserved sequence GAMENWGL (Sequence I.D. No. 2) has yielded products of the expected sizes, about lkb with the 5 1 primer and about 2.5kb with the 3 ' primer. Two closely related but distinguishable 5' sequences designated 012- 1 and 012-2 have been obtained (see Figure 19, Sequence I.D. Nos. 19 and 20) . Over the region above, the deduced sequences for 012-1 and 012-2 are 72% identical. 012-1 AEELRLPSVIPPLLYDLS 012-2 AEELRLPTNIKPLLYNLT PepA AEDLRLPTNIRPLIYDLT 012-1 AEELRLPSVIPPLLYDLS There is 72% identity of the determined amino acid sequence of PepA (Sequence I.D. No. 1) with the sequence deduced from clone 012-1 and 67% identity with the sequence from clone 012-2. The invention will now be described further in the following non-limiting Examples in which: Figure 1 Shows light micrographs of frozen sections of parasitic nematodes stained for microsomal aminopeptidase activities. The activity is revealed by the deposition of reddish blue reaction product seen as a dark band (arrowed) in these black and white photographs; (a) O.ostertagi. ApM-like activity (b) 0.circumcincta ApM-like activity; (c) 0.ostertaσ . ApA- like activity (d) 0. ircumcincta. ApA-like activity. Figure 2 Shows non-denatured 6-16% PAGE stained with L- alanine histochemical reagent for aminopeptidase activity. Track i) HHOD-enriched preparation; ii) Thesit extract of Haemonchus contortus,- iii) Blank; iv) Tween extract of Haemonchus; v) , vi) and vii) PBS, Tween and Thesit extracts respectively of Ostertagia ostertagi the arrow points to ApM-like activity in OoThHS. Figure 3 Shows fractionation of integral membrane proteins from Ostertaσia stertagi ThHS by chromatography on an anion exchange column (Compare Figure 4.); protein absorbance 280 nm, elution 0-100% salt gradient. Figure 4 Shows the distribution of enzyme activities in fractions from anion exchange chromatography shown in Figure 3. Met-pNA, aminopeptidase M-like activity; ApA, aminopeptidase A-like activity. Figure 5 Shows 6-16% SDS-PAGE under reducing conditions of pooled fractions from anion exchange chromatography shown in Figures 3 and 4. i) , v) , molecular weight markers; ii) Fractions 8, 9 and 10; iii) Fractions 11, 12 and 13; iv) Fractions 14, 15 and 16; vi) an H110D- enriched preparation for comparison. Ool2 is arrowed in track ii) . Figure 6 Shows 6-16% SDS-PAGE under reducing conditions of pooled fractions from anion exchange column likely to contain aminopeptidase activity and further fractioned by gel exclusion using superose 12 (mw exclusion of 2 x 10 6 ) . Aminopeptidase activity was only present in Fraction 12, the only fraction with a band at 125,000 (arrowed) . Molecular weight markers and an H110D- enriched fraction were also run for comparison. Figure 7 Shows 6-16% SDS-PAGE under reducing conditions of integral membrane proteins from Ostertaσia circumcincta. Track i) molecular weight markers; ii) proteins soluble in Thesit after pre-extraction with PBS and Tween 20, the band with M r 124,000 (Ocl2) is arrowed. Figure 8 Shows western blots treated with biotinylated lectins a) ϋlex europaeus: b) Triticu vulσaris. c) Concanavalin A: d) pplichPS biflPrUS; of i) H110D- enriched preparation compared with; ii) OcThHS and iii) molecular weight markers. The arrow on (c) indicates Ocl2. Figure 9 Shows fractionation of OcThHS initially passed through an Ulex europaeus lectin column to remove low molecular weight contaminants, by affinity chromatography on a Con A column. Protein absorbance 280 n , Elution 0 to 100% 0.5M sugar (α-methylglucopyranoside) gradient. Figure 10 Shows distribution of enzyme activities in fractions of OcThHS obtained from the Con A column chromatography shown in Figure 9. Met-pNA, Leu-pNA, aminopeptidase M-like activities. Figure 11 Shows 6-16% SDS-PAGE under reducing conditions of fractions from Con A affinity chromatography, initially passed through a Ulex europaeus column. Only the binding fractions with aminopeptidase M-like activity contained a band of Mr 124,000 (arrowed). Figure 12 Shows ion exchange chromatography of pooled fractions from Con A chromatography that bound to the column and which contained aminopeptidase activity; Protein absorbance at 280 nm, salt gradient from 0 to 100%. Figure 13 Shows the distribution of aminopeptidase M-like (Met-pNA) activity in fractions obtained by anion exchange chromatography. Figure 14 Shows pH profiles of the aminopeptidase activity of 0ol2 compared to Hll with leucine p- nitroanilide as substrate. The solid and dashed lines indicate the results obtained for 012 and Hll, respectively. In the assays, MES and MOPS were used to cover the pH ranges 5.5 to 6.5 and 6.5 to 8.0, respectively. Aminopeptidase activity was assayed using 5 μl 012 in PBS/0.1% thesit at a concentration of 0.378 mg/ml and 5 μl Hll in ConA Buffer B at a concentration of 1.25 mg/ml, and assayed over 21 minutes. Figure 15 Shows pH profiles of the aminopeptidase activity of 0ol2 compared to Hll with methionine p- nitroanilide as substrate. The solid and dashed lines indicate the results obtained for 012 and Hll, respectively. The assays were performed as described in the legend to Figure 14 over a period of 13.6 minutes. Figure 16 Shows pH profiles of the aminopeptidase activity of 0ol2 compared to Hll with α-glutamic p- nitroanilide as substrate. The solid and dashed lines indicate the results obtained for 012 and Hll, respectively. The assays were performed as described in the legend to Figure 14 using 5 μl 012 in ConA Buffer A at a concentration of 0.529 mg/ml and 5 μl Hll in ConA Buffer B at a concentration of 2.5 mg/ml over a period of 25 minutes. Figure 17 Shows a sodium periodate treated Western blot of ConA 0ol2 and ConA Hll probed with sheep α-0ol2E serum. Figure 18 Shows the recognition of 0ol2E by serum from sheep injected with 0ol2E, as measured by ELISA. The antiserum titre increased with time after antigen injection illustrating the antigenicity of 0ol2. Antiserum titres against periodate treated antigen were lower than against untreated antigen. The arrow indicates the time of the second injection. Figure 19 Shows the nucleotide sequences of a) 012-1 (Sequence I.D. No. 19) and b) 012-2 (Sequence I.D. No. 20) derived from cloned PCR products. Figure 20 Shows the nucleotide sequences of a) 012-3 (Sequence I.D. No. 21), b) 012-4 (Sequence I.D. No. 22)and c) 012-5 (Sequence I.D. No. 23) derived from cloned PCR products. Figure 21 a(i)-a(v) (Sequence I.D. Nos. 24-28) show the predicted amino acid sequences derived from the DNA sequences 012-1, 012-2, 012-3, 012-4 and 012-5, respectively, shown in Figures 19 and 20 (Sequence I.D. NOS. 19-23) ; b(i)-b(vi) show the predicted amino acid sequences of 012-1 and 012-3 compared with the published amino acid sequences of (i) and (ii) rat microsomal aminopeptidase M (Watt et al. , 1989), (iii) and (iv) mouse microsomal aminopeptidase A (Wu et al. , 1990) and (v) and (vi) Haemonchus Hll-3 respectively; identities are enclosed in boxes, dashes indicate spaces introduced to maximise the level of homology between the compared sequences. The conventional single letter code for amino acids is used. The horizontal line above the sequence indicates the position of the transmembrane region and the asterisks show the position of the zinc- binding motif. Figure 22 Shows the alignments of the amino acid sequence (designated PepA, Sequence I.D. No. l) ) with the predicted amino acid sequences of 012-1 and 012-2. Fiσure 23 Shows the recognition of ConA 0ol2 by serum from cattle injected with ConA 0ol2 (experimental group, squares) or ferritin (control group, circles) , as measured by ELISA. The antiserum titre increased with time after injection of ConA Ool2 but not ferritin, illustrating the antigenicity of 0ol2. The arrows indicates the time of the first, second and third injections of ConA 0ol2 or ferritin. Thirty days after infection with 0. ostertaαi larvae, the control group did not show an increased antiserum titre against ConA 0ol2 illustrating the hidden nature of the antigen in the source worm. Figure 24 Shows a Western blot of ConA 0ol2, ConA Hll and ConA 0cl2 probed with sheep anti-0ol2E (1:1000), bovine anti-ConA 012 (1:1000) and sheep anti-CamQ Hll IgG (1:100) sera. Fiσure 25 shows a 6-16% denaturing, reducing SDS-PAGE gel of chymotrypsin digested PHP separated on ConA sepharose. Lanes 1 and 8 contain molecular weight markers, lane 2 contains starting material, lane 4 contains material which did not bind to the column, lanes 5 and 6 contain material having ApM activity and lane 7 contains 500μg chymotrypsin alone. The arrows indicate bands at approximately 116000. Fiσure 26 shows the release of aminopeptidase activity from Ostertagia ostertaσi after trypsin digestion at various concentrations of the protease. Figure 27 shows a Western blot of ConA 0ol2 after trypsin digestion and gel filtration on Superdex (lane 1) and ConA Hll (lane 2) run on a 6-16% denaturing, reducing gel, probed with α CamQHll IgG at a dilution of 1:100 or 1:1000. The ConA Ool2 fraction from the Superdex fractionation contained ApM activity. Serum used at a concentration of 1:100 was found to cross- react with Ool2. The arrow indicates the band of approximately 116000. Figure 28 shows the histochemical localisation of microsomal aminopeptidase activity within the intestine of Cooperia Oncophora using leucine-SMNA as substrate at a) low power, and b) high power, magnification. Example 1 Histpchemistry O.ostertaσi and O. ircumcincta adults were frozen in embedding compound for frozen sections and 10 μm cryosections were cut on a cryostat. Sections were then assayed using histochemical techniques for ApM-like and ApA-like activity as follows: AMINQPEPTIPASE M Based on methods by Nachlas, Crawford & Seligman (1957, J. Histochemistry and Cytochemistry 5, 264-278) and Lojda, Gossrau & Schiebler (1979, Enzyme Histochemistry. A Laboratory Manual, Springer Verlag, Berlin) . Substrate L-alanine 4-methoxy-β-naphthylamide (Sigma) 0.7mM or L-leucine 4-methoxy-β-naphthylamide (Sigma) 0.7mM Dissolved in absolute ethyl alcohol 0.05 ml Distilled water 0.45 ml 0.1M acetate buffer pH 6.5 5 ml Normal saline (0.85%) 4 ml 0.13% potassium cyanide 0.5 ml Fast Blue B (just before use) 5 mg Method 1. Dry sections at room temperature 2 hr 2. Fix in formal calcium at 4 β C 10 min 3. Rinse in normal saline at 4°C (x2) 2-3 min each 4. Incubate at 37°C in substrate 5-10 min 5. Rinse in normal saline 2 min 6. Rinse in 0.1M copper sulphate 2 min 7. Rinse in saline 2 min 8. Mount in glycerin jelly 9. Inhibit enzyme activity with the addition of 5mM 1,10 phenanthroline to the incubation medium. Enzyme activity appears bluish red. AMINOPEPTIDASE A (Lojda & Gossrau, 1980, Histochemistry 67, 267-290) . Substrate L-Glutamic acid o.-4-methoxy-β-naphthylamide (Sigma) Dissolve in dimethyl formamide 0.1M Phosphate buffer (pH 7.0) Fast Blue B CaCl Warm and filter 1% formalin in 3.5% CaCl Methpd 1. Dry sections at room temperature 2 hr 2. Fix in formal calcium (4°C) 10 min 3. Rinse in phosphate buffer 2 x 2 min 4. Incubate in substrate 10 min 5. Rinse distilled water (4 e C) 10 min 6. Post fix (room temp) 1 hr 7. Rinse distilled water 8. Mount glycerine jelly Enzyme activity appears red. The results are shown in Figure 1, which clearly shows both ApM-like and ApA-like activity associated with the intestinal microvilli of O.ostertagi (Figure 1(a), ApM- like; Figure 1(c), ApA-like) and 0.circumcincta (Figure Kb); ApM-like; Figure 1(d) ApA-like). Example 2 Ostertaσia ostertaσi O.ostertagi was differentially extracted with PBS (to yield PHS extract) , 1% Polyoxyethylene-sorbitan- monolaurate (Tween 20, a Registered trademark of IX+CI Americas Inc, Wilmington, USA) (TwHS extract) and Thesit (ThHS extract) . The extracts were centrifuged at high speed (Beckman Airfuge, maximum speed, angle rotor) and the supernatant liquids were assayed for aminopeptidase M-like enzyme activity (see Table 1) . Aminopeptidase A- like activity is also present in this extract but with a much lower specific and total activity. The ratio of aminopeptidase M (leu/met-pNA) to aminopeptidase A-like (α-glu-pNA) activity was approximately 4:1. The centrifuged O.ostertagi extracts (PHS, TwHS, ThHS) were run on 6-16% non-denaturing PAGE. For comparison HllOD-enriched extracts from Haemonchus contortus were also run on the same gel. Aminopeptidase M activity was demonstrated on the gel using the alanine histochemical substrate. Activity was localised to a single band in the Thesit extract (designated OoThHS) of Ostertagia ostertaαi which migrated the same distance as the slower of two bands having aminopeptidase M activity in the Haemonchus extract (Figure 2) . (A faster moving band was also visualised in the PBS extract of Ostertagia which may be cytoplasmic (non-integral membrane) leucine aminopeptidase) . The centrifuged Thesit extract (OoThHS) waε dialysed in lOmM Tris pH 7.2, 0.1% Thesit and applied to an anion exchange column (Pharmacia: SMART MonoQ column) . The wash-through fractions and those binding fractions released by application of a sodium chloride gradient were collected (Figure 3) . Individual fractions were assayed for aminopeptidase M, and aminopeptidase A-like activities. Most aminopeptidase activity eluted at the start of the salt gradient (Figure 4) . Samples of the fractions were run on a 6- 16% SDS-PAGE under reducing conditions and the separated proteins were visualised by silver staining (Figure 5) . The fractions containing most of the aminopeptidase activity had two particularly strong bands, one with an M r of about 125,000 (arrowed, Figure 5) and a doublet with M r of about 32,000. These components were separated by gel filtration (on a SMART Superose 12 column) , as shown by SDS-PAGE (Figure 6) . Fractions were assayed for aminopeptidase M-like activity. The activity was found only in the fractions containing the band with an M r of 125,000. This protein is designated Ool2. Trypsin digestion of the pelleted material from O.ostertagi obtained after extraction with PBS released aminopeptidase activity in the ratio M/A-like of 8:1. The released protein may be separated by SDS-PAGE, the band excised from the gel and the protein transferred to membrane for N-terminal sequence analysis. Example 3 Ostertaαia circumcincta Homogenates of adult 0.circumcincta were extracted successively in PBS, 1% Tween 20 and 1% Thesit. The extracts were centrifuged at high speed. The centrifuged Thesit extract (OcThHS) contained aminopeptidase M and aminopeptidase A-like activities (Table 2) and as found in O.ostertagi the aminopeptidase A-like specific and total activity is much lower than that of aminopeptidase M. The total aminopeptidase activities are lower than those found in O.ostertagi preparations, A band with M r of 124,000 (Ocl2) is present in OcThHS (Figure 7) . The 0.circumcincta extracts were electroblotted alongside a sample of HllOD and probed with several biotinylated lectins to determine which, if any, would bind to Ocl2. (Figure 8) . Biotinylated Concanavalin A, Ulex europaeus. TritJCUm vulgarJS, Dolichos biflorus and Arachis hypogea lectins were used at dilutions optimised for use with HllOD. As shown in Figure 8, biotinylated ConA lectin binds strongly to Ocl2 (and to many other bands) ; D.biflorus lectin binds very weakly. There was virtually no binding of A. hypoσea and T.vulαar.R lectins. U.europaeus lectin bound to several low molecular weight bands and may be used as an affinity chromatography column for removing low molecular weight protein from OcThHS. The wash-through fractions from such a column were applied to a Con A affinity column and the bound proteins were eluted with a gradient of 0 - 100% 0.5M methylglucoside (Figure 9). Fractions were assayed for aminopeptidase-like activity (Figure 10) . Aminopeptidase M-like activity was present in the binding fractions. When run on 6-16% SDS-PAGE under reducing conditions, only fractions demonstrating aminopeptidase M activity have a band with M r of 124,000 (Figure 11) . The fractions with aminopeptidase activity were pooled and applied to an anion exchange column. The wash-through fractions and fractions released by a salt gradient were collected (Figure 12) . Aliquots were assayed for aminopeptidase M-like activities (Figure 13) . Aminopeptidase M activity was present in the fractions eluted with between 11% and 25% NaCl. Fractions were then run on 6-16% denaturing PAGE, but even after silver staining there was too little protein present to detect clear bands. As indicated for Ostertaσia ostertaσi. the aminopeptidase and proteins may be further purified by gel exclusion chromatography. Table 1. ENZYME ACTIVITIES OF OSTERTAGIA OSTERTAGI EXTRACTS Table 2. ENZYME ACTIVITIES OF OSTERTAGIA CIRCUMCINCTA EXTRACTS Example 4 Characterisation of 0ol2 from 0. ostertaσi The following studies were performed using 0ol2 prepared as described in the following protocol in which Con A buffer A is 5 mM sodium acetate 1 mM manganese chloride, 1 mM anhydrous calcium chloride, 0.1M sodium chloride, 0.02% sodium azide, pH 5.2 and Con A Buffer B is a buffer A with the addition of 0.5M methyl-α-D- glucopyranoside and 0.2M methyl mannoside. 1. Homogenize worms in 10 vols. ice cold PBS + 0.02% sodium azide (PBSa) , centrifuge at low speed (17,000g). 2. Resuspend pellet in 10 vols. PBSa and recentrifuge. Repeat PBSa resuspension and centrifugation once more. PBS low speed supernatant (PLS) can be ultracentrifuged at 100,000g to give a high speed PBS supernatant (PHS) and high speed pellet (PHP) . 3. Resuspend low speed pellet in 10 vols. PBSa + 1% Tween 20 and mix for 1.5 hours at 4°C, spin at 17,000g as above. Repeat washes and spins, twice. 4. Resuspend pellet in PBSa + 1% Thesit and mix overnight at 4°C. Centrifuge as above. Resuspend pellet on PBSa/thesit and mix for 1.5 hours at 4°C, centrifuge and repeat. Thesit low speed supernatants (ThLS 1-3) are pooled and ultracentrifuged at 100,000g to give Thesit High Speed Supernatant (ThHS) . 5. ThHS is buffer exchanged using a Pharmacia PD10 column or dialysed with ConA buffer A + 0.1% thesit and run on a Conconavilin A (ConA) sepharose column. 6. ThHs is applied to the ConA column of either the FPLC or Pharmacia SMART system and unbound protein washed off with ConA buffer A. Bound material is eluted using 100% ConA buffer B, with a pause at the start of elution of 15-20 minutes to maximise the yield of bound protein. Fractions eluted and containing ApM activity (as determined by microtitre plate enzyme assay) are run on 6-16% denaturing, reducing PAGE to ensure the presence of an 012 band and pooled to give ConA 012. 7. ConA 012 can then be dialysed or buffer exchanged into MonoQ buffer A (20mM tris, pH7.2) + 0.1% thesit before application to the Pharmacia MonoQ ion exchange column. Material is loaded onto the column in MonoQ buffer A and eluted using a gradient of 1%/ml MonoQ buffer B (Mono Q buffer A and 1M NaCl) and after reaching 30%, 3%/ml up to 100% buffer B. Eluted fractions containing ApM activity and an 012 band are pooled to give CamQ 012. Inhibitor studies Methpd The effect of various protease inhibitors on 0ol2 activity was assayed and included amastatin (aminopeptidase inhibitor) , actinonin (aminopeptidase inhibitor) , bestatin (aminopeptidase inhibitor) , EDTA (metallo-protease inhibitor) , E64 (cysteine protease inhibitor) , pepstatin (aspartic protease inhibitor) , 1,10 phenanthroline (metallo-protease inhibitor) and 3,4-dichloroisocoumarin (DCI, serine protease inhibitor) . 10 μl Con A 0ol2 and 100 μl 50 mM Mops buffer, pH 7.0 was added to a microtitre plate well and 2-4 μl of enzyme inhibitor added, to give a final concentration of 0.5 mM and 1 mM actinonin, 50 μM and 100 μM bestatin, 1 mM and 15 mM EDTA, 50 μM and 100 μM E64, 10- and 50 μM pepstatin, l mM and 10 mM 1,10 phenanthroline, 100 μM and 500 μM DCI. The plate was preincubatεd at 37°C for 20-28 minutes and then assayed for ApM and ApA activity. 100 μl 2 mM leucine p- nitroanilide (ApM) or α-glutamic p-nitroanilide (ApA) / Mops was added per well and the optical density (OD) at 405 nm read using an Elisa plate reader. The plates were incubated at 37°C for 45-48 minutes and the final OD reading was then taken. The OD change per minute per 10 μl sample was calculated. All assays were carried out in triplicate. Results See Table 3. As expected, only metallo-protease and aminopeptidase inhibitors had any inhibitory effect on aminopeptidase activity with 10 mM phenanthroline giving the greatest inhibition. The differential effects of amastatin on ApM and ApA activity are similar to those described previously for Hll (Graham, et al. , WO 93/23542) , distinguishing Ool2 Ap activity from mammalian Ap. Aoyagi et al. (J. Antibiotics, 43(2): 143-148) demonstrated amastatin as a better inhibitor of pig ApA than of ApM and the opposite is found with Hll and 0ol2. Substrate Specificity MethPd ConA 0ol2 was assayed for enzyme activity in triplicate, on a microtitre plate using a range of substrates. 5 μl of ConA Ool2 (buffer exchanged into PBS/0.1% thesit) or ConA Hll (in ConA buffer B) was added to each well together with 100 μl of 50 mM Mops pH 7.0 (or 90 mM citrate buffer + 10 mM NaCl, pH 5.0 for acid phosphatase and 50 mM Tris, pH 9.0 for γ-GTP. The plate was preincubated for 15 minutes and then 100 μl 2 mM substrate added (in the appropriate buffer) before the OD.was measured. The plate was incubated at 37°C for 26 minutes and the OD measured again. For acid phosphatase, the reaction was stopped by adding 75 μl 1.66M NaOH/well. The ratio of enzyme activity was calculated with methionine taken as having an OD of 1. Results See Table 4. ConA 0ol2 shows greatest specificity for methionine and the greatest difference between ConA 0ol2 and ConA Hll is the lower specificity of 0ol2 for lysine and very low γ-GTP activity. pH Optima of Qol2 Ap Activity Method ConA 0ol2 in either ConA buffer A/0.01% Thesit or PBS/0.1% Thesit was assayed for aminopeptidase activity using either a-glutamic-pNa (ApA substrate) , ethionine- pNa or leucine-pNA (ApM substrates) . 50 mM MES (pH 5.5- 6.5) and 50 mM MOPS (pH 6.5-8.0) were chosen as buffers, covering a broad pH range and overlapping at pH 6.5. Buffers were prepared using volumetric flasks and MilliQ ultra pure water and the assays were carried out in triplicate on microtitre plates (as described above) . 5 μl of ConA Ool2 or 5 μl ConA Hll (in ConA Buffer B, for comparison) were added per well and the OD change was measured over 13.6-25 minutes. Results See Figures 14-16. Differences in the pH profiles between substrates and between 012 and Hll are found, although generally the optimum pH for Aminopeptidase activity is between 6.5-7.0. There is also some variability in activity when using MOPS and MES at pH 6.5, indicating that buffer composition also influences activity. Table 3 CON ' A 012 (00) AMINOPEPTIDASE INHIBITION ASSAYS I σ. i Assays carried out in 50mM Mops, pH 7.0 except for AcP (90mM citrate buffer, pH 5.0) and γGT^ (50 mM Tris, pH 9.0) . Assays carried out over 26 min. Example 5 Polymerase chain reactions Preparation of mRNA 80 mg. of 12 day old Ostertagia ostertagi (UK strain) flash frozen and stored in liquid nitrogen were ground to a power under liquid nitrogen and the mRNA extracted using a micro mRNA extraction kit (Pharmacia) according to the manufacturer's instructions. Briefly, the powdered nematodes were solubilised in a solution containing guanidinium thiocyanate and N-lauroyl sarcosine and then the mRNA extracted from the aqueous solution with oligo (dT)-cellulose. The mRNA was stored under ethanol at -80°C until use. Preparation of First Strand cDNA mRNA, from the equivalent of 10 mg. of O. ostertagi. as prepared above was used for the preparation of first strand cDNA. The mRNA was dissolved in 11.5 μl of sterile water and 1 μl of a T17 adaptor-primer, 0.5 μg/ μl, (5 1 GACTCGAGTCGACΑTCGATTTTTTTTTTTTTTTTT 3") added. This mixture was heated to 65°C for 10 minutes, left at room temperature for 2 minutes, then placed on ice. To synthesize cDNA the following components from a cDNA cycle kit (Invitrogen) were added, RNase Inhibitor to 0.5 units/μl, Reverse transcriptase buffer to lx concentration, dATP, dGTP, dCTP and dTTP to 1.25 mM, Sodium pyrophosphate to 4 mM and AMV Reverse Transcriptase to 0.5 units/μl (all given as final concentrations) . The reaction mix was incubated at 42°C for 65 minutes and then heated at 98°C for 2 minutes. The reaction mix was diluted by addition of 100 μl water, then extracted with phenol and the cDNA purified by spun column chromatography. (Maniatis et al f 1982) . The cDNA was stored at 4°C. 39 - PCR Amplification of the cDNA using Aminopentidase- Specific Primers The Primers DNA sequence data from the nematodes H. contortus (WO 93/23542) and Caenorhabditis eleσans with homology to mammalian aminopeptidase were compared and a conserved region coding for the amino acids: Gly Ala Met Glu Asn Trp Glu Leu, was obtained. Both sense and anti-sense primers were manufactured based on this sequence. The sense primer was 5' GGA/TGCIATGGAA/G AAT/C TGGGGICT 3' (Sequence I.D. No. 3) and the anti-sense 5' AGICCCCAA/G TTT/C TCCATIGCT/A CC 3' (Sequence I.D. No. 4). The sense primer described above can be used with the adaptor sequence from the T17 adaptor-primer, 5 1 GACTCGAGTCGACATCGA 3 ! , to PCR with the first strand cDNA from O. ostertagi. Some 5' ends of cDNAs of various helminths have been shown to have a conserved region of 22 base pairs before the initiating methionine (Huang 1990) . This region, known as Spliced leader 1(SL1), has the sequence 5' GGTTTAATTACCCAAGTTTGAG 3' (Sequence I.D. No. 5). This spliced leader has been identified at the 5' end of microsomal aminopeptidase of H. contortus (WO 93/23542) . This spliced leader paired with the anti-sense primer described above can be used to perform PCR with the first strand cDNA from 0. ostertagi. Polymerase Chain Reactions The reactions were carried out using a Programmable Thermal Cycler (M.J. Research Inc) . The first reaction mix contained 1 μl of O. ostertagi first strand cDNA, lpmol of T17 adaptor-primer, 25 pmol of the adaptor, 25pmol of the sense primer, lx Tag buffer (Boehringer Mannheim), 0.2 mM each of dATP, dCTP, dGTP and dTTP, in a reaction volume of 100 μl. The second reaction.mix contained 1 μl of cDNA , 25 pmol of SL1 primer, 25 pmol of the anti-sense primer and then as for the first reaction mix. The reaction mixes were heated to 95°C for 2 minutes and then held at 80°C whilst 3 units of Tag Polymerase (Boehringer Mannheim) were added to each. The following programme was then performed: Step 1 53° or 56°C for 5 minutes Step 2 72°C for 20 minutes Step 3 94°C for 45 seconds Step 4 53° or 56°C for 2 minutes Step 5 72°C for 2.5 minutes Step 6 39 times to Step 3 Step 7 72°C for 15 minutes Step 8 Hold at 4°C. Purification and Cloning of PCR PRoducts The reaction products were separated by electrophoresis through a 1% agarose gel in TAE buffer (Maniatis 1982) . The banding pattern was the same for reactions at 53°C or 56°C for both primer combinations and so the reaction products for each pair of primers were mixed. These combined reaction products were electrophoresed on a TAE gel and bands of interest were excised and the DNA purified with glassmilk (Geneclean, BIO 101) . The purified DNAs were ligated into the plasmid vector pT7Blue (Novagen) and then used to transform competent Novablue (Novagen) E. coli cells. The transformed E. coli were plated out to allow colour selection of colonies containing an insert plus the vector. DNA from putative positive colonies was prepared and electrophoresed in a TBE gel (Maniatis 1982) , to allow selection of clones with an insert of the desired size. Sequencing pf Clpnes Clones were sequences by 'oligonucleotide walking' of the double-stranded DNA with T7 DNA polymerase using GTP and ITP labelling from a Sequenase 2.0 Sequencing kit (Amersham) . Example 6 Ool2E Sheep Antiserum 1.242 mg ConA 0ol2 was run on a 0.7 mm 8% denaturing, reducing PAGE, adjacent to high molecular weight protein standards. Thin strips were cut from either side of the gel and stained with Coomassie blue to locate the 012 band. These strips were then re-aligned with the gel and the unstained 012 band position estimated and cut out. The gel was homogenised and mixed with Freund's adjuvant and injected into a single sheep, to raise antibodies to 0ol2E (0ol2 - after electrophoresis) . A second injection was administered after 4 weeks using the above method and with the amount of protein run on the gel increased to 1.863 mg. Blood was collected from the sheep, and serum prepared, at regular intervals. The α-0ol2E serum was used to probe 10 mM sodium periodate treated western blots of ConA 0ol2 and ConA Hll. ConA 0ol2 demonstrated strongly immunoreactive bands at about 120,000 and 170,000 and slightly weaker bands at 97 and 55 kDa (Figure 17) . In ConA Hll an immunoreactive band was present at about 70 kDa with Hll appearing very faint. The appearance of an immunoreactive band at 170,000 kDa in ConA 012 requires further investigation. Serum antibody titres were also measured using the Elisa method, Dynatech Immulon 4 Elisa plates were coated with 50 μl/well 3 μg/ml ConA Ool2 at 4°C overnight. Plates were washed in 3x PBS and blocked for 1 hour at 37°C in PBS/10% foetal calf serum (FCS) . Half the plate was treated with 150 μl/well 10 mM sodium metaperiodate in 0.1M sodium acetate buffer pH 5.5 for 30 minutes and then 150 μl 50 mM sodium borohydrate in PBS for 30 minutes at room temperature. The other half of the plate was treated with acetate buffer then PBS alone. Wells were rinsed 3x PBS and 100 μl/well primary antiserum (serum from the sheep pre- and post-injection) diluted 1:1000 in PBS/10% FCS added. Each serum was assayed in triplicate and applied to both sodium metaperiodate treated and untreated wells. The plate was incubated for 1 hour at 37°C, rinsed 3x PBS/0.05% Tween 20 and 50 μl/well donkey a-sheep IgG peroxidase conjugate added, diluted 1:1000 in PBS/10% FCS. The plate was incubated at 37°C for 1 hour, rinsed 3xPBS/ Tween and developed using Tetramethyl Benzidine (TMB) by the addition of 150 μl/well of the following developing solution: 18 ml MilliQ water, 2 ml 1M sodium acetate pH 6.0, 200 μl TMB (10 mg/ml in DMSO) and 20 μl 6% H 2 0 2 . Development was left for 10 minutes and stopped with 30 μl/well 3M H 2 S0 4 . The OD was read on an ELISA plate reader at 450 nm. Results See Figure 18. Ool2E is antigenic in the sheep with antiserum titres rising with time. Sodium periodate treatment of the antigen results in decreased OD'ε suggesting that the majority of the immune response is directed towards carbohydrate and less so to protein epitopes. Example 7 Con A 0ol2 Bovine Antiserum Con A 0ol2 was prepared from thesit extracted 0. ostertagia according to the protocol given at the beginning of Example 4. 6 calves, aged between 12 and 27 weeks old, were given 3 injections of ConA 0ol2 in Freund's adjuvant. 250 μg/calf was administered for the first two doses and 200 μg in the final injection with injections given at 3 week intervals. 6 calves in the control group were each given three injections of 150 μg ferritin in Freund's adjuvant (Type 1, from horse spleen) . Calves were then challenged with 40000 infective O. ostertagi larvae. Serum was prepared from samples of blood collected weekly and serum antibody titres were measured using the Elisa method as follows. Dynatech Immulon 4 Elisa plates were coated with 50 μl/well 3 μg/ml ConA Ool2 at 4°C overnight. Plates were washed in 3X PBS and blocked for 1 hour at 37°C in PBS/5' foetal calf serum (FCS) . Wells were rinsed 3X with PBS/0.05% Tween 20 and 100 μl/well primary antiserum diluted 1:1000 in PBS/5% FCS added. Each serum was assayed in triplicate. The plate was incubated for 1 hour at 37°C, rinsed 3X with PBS/0.05% Tween and 50 μl/well rabbit α-bovine IgG peroxidase conjugate added, diluted 1:1000 in PBS/5% FCS. The plate was incubated at 37°C for 1 hour, rinsed 3X with PBS/Tween and developed using tetramethyl Benzidine (TMB) by the addition of 150 μl/well of the following developing solution: 18ml MilliQ water, 2ml 1M sodium acetate pH 6.0, 200 μl TMB (lOmg/ml in DMSO) and 20 μl 6% H 2 0 2 . Development was left for 15 minutes and stopped with 30 μl/well 3M H 2 S0 4 . The OD was read at 450 nm. Results See Figure 23. Con A 0ol2 is antigenic in cattle with antiserum titres rising with time. Example 8 I munpreactivity oi Bpvine and Sheep Q12 anti-serum ConA Ocl2 was prepared from 0. Circumcincta using the standard thesit extraction protcol with a freshly packed ConA sepharose column and bound material was eluted from the column with 0.5M methylglucopyranoside and 0.2M methylmannoside in ConA buffer. ConA 0cl2 was run on two 6-16% denaturing, reducing gels together with ConA 0ol2 and ConA Hll. About 4μg of each preparation was run per lane. The proteins were then western blotted onto Immobilon P and probed with the following sera: sheep anti-CamQ Hll IgG (1:100), sheep anti-Ool2E (as prepared in Example 6, 1:1000), bovine anti-ConA Ool2 sera (as prepared in Example 7, 1:1000) and pre-immune sheep and bovine sera (1:1000) as negative controls. Results See Figure 24. ConA Hll, ConA Ool2 and ConA 0cl2 demonstrated immunoreactivity to all three immune sera. 0ol2 appeared smaller than 0cl2, which may have resulted from degradation of the former. Ocl2 cross-reacts with antisera to 0ol2 and together with the evidence that 0ol2 elicits an immune response in sheep, this suggests that Ool2 may have utility as a bivalent vaccine, acting against 0. circumcincta as well the source worm. Example 9 Proteolytic digestion of 012 Protease Digestion of Ool2 PhP Method: Oo PhP was resuεpended in 50mM Mops buffer, pH 7.0 to give O.lmg pellet/ml. 50 μl aliquots were prewarmed at 37°C for 39 minutes. 5 μl of either α- Chymotrypsin (Sigma type VII, 50 units/mg solid) , Subtilisin (Sigma type VIII, 11.6 units/mg solid), Trypsin (Sigma, TPCK-treated type XIII, 9700 units/mg solid) or Thermolysin (Sigma, 37 units/mg solid dissolved in lmM HCl/2mM CaCl) was added to give final protease concentrations of 5, 50, 500, 1000 and 2000 μg/ml. Aliquots containing chymotrypsin, trypsin or subtilisin were then incubated at 37°C for 1 hour and protease activity inhibited by the addition of 1 μl lOmM 3,4-dichloroisocoumarin (DCI). Aliquots containing thermolysin were incubated for 2 hours at 37°C and activity inhibited by the addition of 1 μl 0.25M EDTA. Samples were spun at high speed on the airfuge at 20 psi for 20 minutes at room temperature. 5μl samples were assay for ApM activity, using the microtitre plate method, over 29.6 minutes with methionine-pNA as substrate. Results See Table 5. All four proteases released ApM from the PhP, but thermolysin proved to be the least effective. Chymotrypsin Digestion of PHP The above experiment was performed on a larger scale to further purify the released ApM activity. Oo PHP 2 and 3 were resuspended in 50 mM Mops pH 8.0 to give O.lmg pellet/ml and 4.5ml was prewarmed at 37°C for 20 minutes. The digestion was carried out as above with α-chymotrypsin added to give 500 μg protease/ml suspended PHP. Protease activity was inhibited by the addition of 15 μl/ml 10 mM DCI. The digests were then spun twice on a Beckman ultracentrifuge using a SW60 swing-out rotor at 31000 rpm for 1 hour 20 minutes. Enzyme assays demonstrated the release of ApM and ApA activity from the PHP (0.734 nmoles/min/lOμl with Met- pNA; 0.414 nmoles/min/10 μl with Leu-pNA and 0.304 nmoles/min/10 μl with o_-Glu-pNA) . The remaining supernatant was buffer exchanged using a Pharmacia PD10 column into ConA Buffer A and this was loaded onto a freshly packed ConA sepharose column for the SMART system. Material was loaded onto the column in ConA buffer A and eluted in one step using 100% ConA buffer B containing 0.5M methylglucopyranoside and 0.2M methylmannoside. Only eluted fractions contained ApM activity and these were pooled and run on 6-16% PAGE. A strong band was apparent at around 116000 in these fractions (Figure 25) . Pooled fractions were concentrated in a centricon 10 and run on a 6-16% denaturing, reducing minigel together with undigested PHP, chymotrypsin only, ConA Ool2 and ConA Hll for comparison. This was western blotted (using the BioRad semi-dry system) onto Immobilon P, treated with lOmM sodium metaperiodate and probed with Sheep o_-Ool2E (1:1000) . A strongly immunoreactive band was apparent at about 116000-120000 in the digested pellet. Supernatant from digested PHP was run on a second gel using tank buffer containing 0.192M tricine instead of glycine. The gel was blotted and probed as above and the immunoreactive band excised from the blot and the amino acid sequence analysed. No matches to any known sequences were found. Trypsin digestipn p£ 012 The residue obtained by centrifuging an homogenate of Q. ostertaαi in phosphate-buffered saline containing 0.02% sodium azide (PBSa) at 26,500g (max) for 20 minutes in a swing-out rotor was re-extracted 3 times with PBSa, once with 1% polyoxyethylenesorbitan monolaurate (Tween 20) in PBSa and once with 50 mM 3- [N-morpholino]propane- sulphonic acid (MOPS) pH 8.0. The extracted residue was resuspended in 50 mM MOPS to give the equivalent of 0.17 g worm starting material per ml. Aliquots were digested with 0.5, 5, 50 or 500 μg TPCK-trypsin (trypsin treated with L-1-tosylamide-2-phenylethylchloromethyl ketone)/ml. Digestions were stopped by the addition of 10 mM DCI (15 μl/ml) . Treatment with 50 μg TPCK-trypsin (485 units) released the greatest amount of microsomal aminopeptidase M activity (Figure 26) . Release of microsomal aminopeptidase A activity was negligible from pellets digested with 0-50 μg TPCK-trypsin/ml. In another experiment material from 5g O. ostertaαi was digested twice with 50 μg/ml TPCK-trypsin. About 33% of the original amount of ApM waε released after a second digest. Both digests were pooled and subjected to affinity chromatography on Concanavilin A. Microsomal aminopeptidase M activity bound to the column, but only 32% of the applied activity was eluted with 0.5M methylglucopyranoside. Fractions were run on SDS-PAGE on a 6-16% inigel. After Coomassie blue staining, a band located around the 116,000 region, about the size expected from tryptic digestion of 012 was located in the fractions having the microsomal aminopeptidase activity. (Amino acid sequences from this and the adjacent bands did not match any sequence in the data bases.) A further 4.3g of 0. ostertagi was digested twice with TPCK-trypsin as above. The total aminopeptidase activity solubilised equalled 455 nmoles/min (Table 6) . The high speed supernatants from the first and second digests (Trypsin HS1 & HS2) were buffer exchanged with ConA buffer A and run on a ConA sepharose column. The start material applied to the column was enriched for ApM activity, having a ratio of ApM:ApA of 18:1. All the ApM activity bound to the column (there was none in the wash-through) ,- and 67% of this was recovered by elution with 0.5M methylglucopyranoside + 0.2M methylmannoside. (Thuε the use of two sugars in the eluting buffer more than doubled ApM recovery from the ConA column.) Fractions containing ApM activity were pooled, concentrated and subjected to gel filtration (on an FPLC Superdex 75 HR10/30 (Pharmacia) column) . Fractions were concentrated, assayed for ApM activity and run on 6-16% SDS PAGE. Several protein bands were present. After Western blotting and probing with anti- CamQHll antibody, a band at between 110,000-116,000 proved immunoreactive (Figure 27) . ConA and Superdex fractions were re-run on a 6-16% reducing/ denaturing minigel, with tank buffer containing 0.192 M tricine instead of glycine and electroblotted onto Problott. Several bands were cut out and sequenced (but did not yield sequences which matched any sequence in the data bases.) The sequences obtained after proteolytic digestion are as follows: EPLAPGAANP KGVANDTMQF (Sequence I.D. No. 6) DXEEEXQPEN GXRTRGRXPX (Sequence I.D. No. 7) DEXCAVPEEA XGVDXPVXGE (Sequence I.D. No. 8) XVFPGTEYGA LLQFAQANAQ RQNLR (Sequence I.D. No. 9) DVPGADDDNG QDNDGEFVGE (Sequence I.D. No. 10) APFEDDAPPA GGPKGLLEXL SAGLA (Sequence I.D. No. 11) LQNQLPAF (Sequence I.D. No. 12) KXDPGDGNYG GGXGGFQGHL (Sequence I.D. No. 13) SIPNGDTGPX XNGYXAENYN QGGXE (Sequence I.D. No. 14) DXEPGPREPG GSEGERQPEE G (Sequence I.D. No. 15) XAEDLRLPTN IRPLIYDLTR (Sequence I.D. No. 16) RGWXPVDXX QTPVPGQPVN (Sequence I.D. No. 17) XXQGSPYDXX (Sequence I.D. No. 18) ApM activity has been successfully extracted from pellets recovered from ultracentrifuged PBSa and 1% Tween 20 extracts of 0. ostertaαi using either 1% thesit or 500 μg TPCK-trypsin/0.15g pellet. Trypsin digests were western blotted probed with anti-CamQHll IgG and immunoreactive bands cut out for amino acid sequencing, however no usable sequence could be determined. Table 5 AMINOPEPTIDASE M ACTIVITY RELEASED BY PROTEASE DIGESTION OF OSTERTAGIA OSTERTAGI PHP Table 6 AMINOPEPTIDASE ACTIVITY FROM TRYPSIN DIGESTED OSTERTAGIA OSTERTAGI I Example IQ ApM Localisation in Cooperia oncophora 10 μm frozen sections of Cooperia oncophora were stained histochemically for ApM activity using leucine-βMNA (as performed in Example 1) . ApM activity was found to be localised to the intestinal microvilli (see Figure 28) . Example 11 Antibody response to in-iection of 012 and vaccination trial in cattle Twelve worm-naive calves were housed indoors and were allocated to 2 groups, C and E, each comprising 6 calves matched for age, breed and sex. All calves received a primary vaccination on day -63. Those in group E were vaccinated with 250 μg ConA 0ol2; those in group C with 150 μg horse spleen ferritin (purchased from Sigma Chemical Co. Ltd., Poole, Dorset, UK) in order to act as negative (adjuvant) controls. Primary immunisation was with aluminium hydroxide and Freund's Complete adjuvant (FCA) . Twenty one days later (day -42) all calves received a secondary vaccination, with a tertiary vaccination being administered after a further 21 days (both of 200μg ConA 0ol2 or 150μg ferritin) , both using Freund's incomplete adjuvant (FIA) . On day 0 all calves received an exogenous challenge of ca 40,000 L3 larvae of 0. ostertagi. The levels of nematode infection were monitored by faecal egg counts, and determination of worm burden at necropsy twenty seven to twenty eight days post challenge. Results The mean worm burden of calves vaccinated with 012 was reduced relative to control animals vaccinated with horse spleen ferritin. Example 12 Inhibition of Q12 microsomal aminoOeotidase activity hy bovine serum containing antibodies to ConA 0ol2 An enyzme assay was carried out using the ELISA plate method (Example 7) to determine the effect of i) pre- immune, ii) anti-ferritin (control) and iii) bovine anti-ConA 0ol2 (immune) sera on the microsomal aminopeptidase activity of ConA 0ol2 in vitro. The pre- immune serum (i) was the pooled serum taken from the experimental group of calves (Group E, Example 7) 70 days pre-infection, the anti-ferritin serum (ii) was the pooled serum taken from the control group of calves (Group C, Example 7) and the bovine anti-ConA Ool2 serum (iii) was the pooled serum taken from the experimental group of calves (Group E, Example 7) at 27/28 days post infection. 10 μl ConA Ool2 (337 μg/ml) and 70 μl 50mM MOPS buffer, pH 7.0 was added per well of a microtitre plate. 20μl serum was added per well to bring the total volume per well to 100 μl. Control incubations included a buffer blank (MOPS buffer only) and ConA Ool2 without the addition of serum. The plate was preincubated for 30 minutes at 37°C then 100 μl/well 2mM methionine p- nitroanilide or leucine p-nitroanilide in MOPS buffer were added. The OD was read on an Elisa plate reader at 405nm and the plate incubated for a further 30 min at 37°C. The OD was read again and the change in absorbance and percentage reduction in microsomal aminopeptidase activity calculated. Results Sample % Reduction in ApM Activity Methionine-pNA as substrate ConA0ol2 + 20μl preimmune serum (group E) 8.0 ConAOol2 + 20μl control serum (group C) 21.4 ConA0ol2 + 20μl immune serum (group E) 41.7 Leucine-pNA as substrate ConA0ol2 + 20μl preimmune serum (group E) 17.1 ConAOol2 + 20μl control serum (group C) 18.3 ConA0ol2 + 20μl immune serum (group E) 40.2 The serum from calves vaccinated with ConA 0ol2 inhibited the aminopeptidase activity of 012 significantly compared to serum from the calves prior to vaccination (Pre-immune serum) , or serum from control calves injected with horse spleen ferritin. SEQUENCE LISTING ( 1) GENERAL INFORMATION : ( i) APPLICANT: (A) NAME: Biotechnology and Biological Sciences Research Council (B) STREET: Babraham Hall, Babraham (C) CITY: Cambridge (E) COUNTRY: United Kingdom (F) POSTAL CODE (ZIP) : CB2 4AT (A) NAME: Danielle McMichael-Phillips (B) STREET: 6 Mingle Lane, Great Shelford (C) CITY: Cambridge (E) COUNTRY: United Kingdom (F) POSTAL CODE (ZIP) : CB2 5BQ (A) NAME: Edward Albert Munn (B) STREET: 72 Station Road, Fulboum (C) CITY: Cambridge (E) COUNTRY: United Kingdom (F) POSTAL CODE (ZIP) : CB1 5ES (A) NAME: Margaret Graham (B) STREET: 17 Bawtree Crescent, Linton (C) CITY: Cambridge (E) COUNTRY: United Kingdom (F) POSTAL CODE (ZIP) : CB1 6XQ (ii) TITLE OF INVENTION: Vaccines (iii) NUMBER OF SEQUENCES: 28 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.25 (EPO) (v) CURRENT APPLICATION DATA: APPLICATION NUMBER: WO GB94/ (vi) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: GB 9322702.3 (B) FILING DATE: 03-NOV-1993 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: Ala Glu Asp Leu Arg Leu Pro Thr Asn lie Arg Pro Leu lie Tyr Asp 1 5 10 15 Leu Thr (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Gly Ala Met Glu Asn Trp Gly Leu 1 5 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: GGWGCNATGG ARAAYTGGGG NCT 23 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: AGNCCCCART TYTCCATNGC WCC 23 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: GGTTTAATTA CCCAAGTTTG AG 22 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Glu Pro Leu Ala Pro Gly Ala Ala Asn Pro Lys Gly Val Ala Asn Asp 1 5 10 15 Thr Met Gin Phe 20 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: Asp Xaa Glu Glu Glu Xaa Gin Pro Glu Asn Gly Xaa Arg Thr Arg Gly 1 5 10 15 Arg Xaa Pro Xaa 20 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Asp Glu Xaa Cys Ala Val Pro Glu Glu Ala Xaa Gly Val Asp Xaa Pro 1 5 10 15 Val Xaa Gly Glu 20 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: Xaa Val Phe Pro Gly Thr Glu Tyr Gly Ala Leu Leu Gin Phe Ala Gin 1 5 10 15 Ala Asn Ala Gin Arg Gin Asn Leu Arg 20 25 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide ( i) SEQUENCE DESCRIPTION: SEQ ID NO: 10: Asp Val Pro Gly Ala Asp Asp Asp Asn Gly Gin Asp Asn Asp Gly Glu 1 5 10 15 Phe Val Gly Glu 20 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: Ala Pro Phe Glu Asp Asp Ala Pro Pro Ala Gly Gly Pro Lys Gly Leu 1 5 10 15 Leu Glu Xaa Leu Ser Ala Gly Leu Ala 20 25 (2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: 1inear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: Leu Gin Asn Gin Leu Pro Ala Phe 1 5 (2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: Lys Xaa Asp Pro Gly Asp Gly Asn Tyr Gly Gly Gly Xaa Gly Gly Phe 1 5 10 15 Gin Gly His Leu 20 (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: Ser lie Pro Asn Gly Asp Thr Gly Pro Xaa Xaa Asn Gly Tyr Xaa Ala 1 5 10 15 Glu Asn Tyr Asn Gin Gly Gly Xaa Glu 20 25 (2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: Asp Xaa Glu Pro Gly Pro Arg Glu Pro Gly Gly Ser Glu Gly Glu Arg 1 5 10 15 Gin Pro Glu Glu Gly 20 (2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: Xaa Ala Glu Asp Leu Arg Leu Pro Thr Asn lie Arg Pro Leu lie Tyr 1 5 10 15 Asp Leu Thr Arg 20 (2) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: Arg Gly Val Val Xaa Pro Val Asp Xaa Xaa Gin Thr Pro Val Pro Gly 1 5 10 15 Gin Pro Val Asn 20 (2) INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: Xaa Xaa Gin Gly Ser Pro Tyr Asp Xaa Xaa 1 5 10 (2) INFORMATION FOR SEQ ID NO: 19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1062 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: GGTTTAATTA CCCAAGTTTG AGATGACAGC ACAATGGGCT AAAAGGACAG TGTTACGGTT 60 TACGCCCATC AGCCTTCTCG TCGCTTCATT AGCAATGGCT CTAGCAATCG CTCTCTCCAT 120 AGGTCTCACT TATCACTTCG TGCGAAATGC TTATGACACT TCACACAATG GAAAGGATCA 180 CCCTGGAGGT GATGACAATT CTCCTTCTGC AGAAGAACTA CGTCTTCCGA GAAGCGTAAT 240 ACCGTTGCTA TACGACTTGA GCATCAAAAC GTATCTGCCC AATTACGTGG ATTTCCCGCC 300 AGAGAAGAAT CTCACCTTTG ATGGGCAAGT GGAAATCTCC ATGGTAGTGA TGGAACCAAC 360 TAGAAGTATT GTACTAAATG CGAAGAATAT TACTGTGATA CCAGAGAAAT GCGAGGTGTT 420 TTCGGGCGAT GAAAAACTGG AAATTGAAAG TGTGATGGAG CATGAGCGGC TTGAGAAGCT 480 GGAATTCCTG TTGAAGAAGC GGTTAGAAAA AGATCAAAAA GTTCTGCTCA AGGTAATCTA 540 CATCGGCCTT ATTAGCAACA CCCTTGGTGG ACTGTACCAA GCTACTTATA CACACACGGA 600 TGGAACCACC AAAATTGCTG CAGCTTCTCA AATGGAACCA ACAGATGCCC GTCGAATGGT 660 GCCATGTCTA GACGAACCCA ACTATAAAGC TAATTGGACA GTCACTGTGA TTCACCCGAA 720 AGGCACAAGG GCCGTATCGA ATGGTATTGA AACGAACGGC GAAGGGGATA TCAATGGCGA 780 CTGGATCATA TCGAAGTTCG AAACCACTCC ACGGATGTCC TCCTATCTAC TGGCAGTTAT 840 TGTCTCGGAA TTCGACTTCA TCGAAGGCAA CACGACAAGT GGTGTGCGGT TCCGAATATG 900 GTCACGCCCT GAAGCGAAGA ATATGACACA ATATGCCTTA GACGCCGGCA TCAGATGTTT 960 AGAGTTTTAC GAGAGCTTCT TCGGCATAAA ATTCCCTTTA CAAAAGCAAG ATATGGTTGC 1020 GCTTCCTGAC TTCTCTGCAG GAGCCATGGA AAACTGGGGC CT 1062 (2) INFORMATION FOR SEQ ID NO: 20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1074 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20: GGTTTAATTA CCCAAGTTTG AGATGGAGGA GCCGAGGGCA CGGAGGCGGA CAGTGCTACG 60 GCTCACGCCC ATCACCCTTT CGATCGCTTT GTTAGGCATA GCAGTTGCCG TTGCGCTCTC 120 TATTGGTCTC ACCTATCTCT TCACACGAAA TGCCTACGAT ACGTCGCGGA AACCGAAGGA 180 ACCAGATCAC CCCGGAGGCG GGGATGACAA TCCTCCTTCT GCAGAAGAAT TACGTCTCCC 240 CACAAACATA AAGCCGTTAC TGTACAATTT GACCATCAAA ACGTATCTGC CCGGTTACGT 300 GGATTTCCCA TCGGAGAAGA ATCTCACCTT CGATGCTCAA GTGCTAATTT CCATGGTGGT 360 AGTGGAGCCA ACCAAAAGTA TCGTGCTGAA TGCCAAGAAA ATCACGGCGC TACCGAAAGA 420 ATGCGAGGTG TTCGCAGGCG ATCAAAAACT GGACATTGAA AGTGTGACGG ATCATGAAAG 480 GCTGGAGAAG CTGGAGTTCA CTCTGAAGCA ACAACTAGAA AAAGACCAGA AAATCCAGCT 540 CAAGGTTGTC TATAGCGGCC TCATTAGCGA CACCCTTGGT GGACTGTATC AAGCTACTTA 600 CAAAGACACG GATGGAACGA CCAAAATCGT CGCAGTCTCT CAAATGGAGC CAACAGACGC 660 TCGTCGAATG GTGCCGTGTT TTGACGAGCC GAGTTTCAAA GCCAACTGGA CAGTAACAAT 720 AATTCATCCA AGAGGTACAA CAGCCGCATC GAATGGCATA GAAACTAACG GTGAAGGAGA 780 ACCCGACGGT GATTGGATCA CATCAAAATT CAAAACCACT CCACGAATGT CTTCTTATCT 840 GCTGGCCGTT ATTGTCTCAG AATTCAAATA TATTGAAGGG CGCACGAAAA GCGGTGTGCG 900 GTTCCGAATA TGGTCACGTC CAGAGGCGGT AAAAATGACG AAATTCGCGT TAGACGCCGG 960 TGTCAGATGT TTGGAATTCT ATGAAAAGTT TTTCGACATT CGATTTCCTC TTGAGAAGCA 1020 AGATATGGTT GCTCTTCCTG ATTTCTCAGC AGGAGCCATG GAGAACTGGG GCCT 1074 (2) INFORMATION FOR SEQ ID NO: 21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1848 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: GGTGCGATGG AGAATTGGGG GCTCATCACT TACAGAGAGA ATTCCTTGTT GTACGATGAA 60 AGATACTATG CACCGTTGAA CAAGGAACGG GTTGCCATCG TGGTTGCTCA TGAACTTGCC 120 CATCAGTGGT TCGGTAATCT TGTCACGTTG AAGTGGTGGG ATGATTTGTG GCTGAACGAA 180 GGTTTCGCAA CATTTGTTGA ATTCATCGGT GCAGATCATA TCAGCAATGG AACCTTTAGA 240 ATGAAGGACT ATTTTCGATT GGATGCACTT GTAGACGCCT TGGAGGCTGA TGCGGTAGCT 300 TCAAGCCATC CGCTATCATT CAAAATCGAT AAAGCTTCAG AAGTTTACGA AGCTTTCGAC 360 GCTATCACGT ACTCCAAAGG AGCGTCAGTT CTCACAATGT TGCAAGCGTT GATTGGTGAA 420 GAAAATTTCA AACAAGCCGT AACGCAATAC CTCAATAAGT TTTCGTTCGA CAACGCGAAA 480 GCGTCCGATC TTTGGGGTGT CTTCGATGAA GTTGTTAAGG ACGTCAAGGG CCCCGACGGT 540 AATCCTATGA AAACCACTGA ATTCGCTTAT CAGTGGACGA CTCAGATGGG CTACCCAATA 600 GTCACAGTGG AAACGTTCAA CGCAACTTCT TTGAAAGTCT CACAAAACCG ATACAAGACG 660 AATAAGGACG CCCAGGAGCC GGAAAAGTAC CGTCATCCAA AATATGGGTT CAAGTGGGAT 720 GTTCCTTTAT GGTATCAAGA AGGCGAAAGC AACGAAATAA AGCAGACCTG GTTGACCAGA 780 GGCGAGCCCC TTTATTTGCA CGTGAGCAGT TCTGATGATT CCATTGTGGT GAACGCCGAT 840 CGGCATGGAT TTTACAGGCA AAACTACGAT GCCAACGGTT GGCGAAAGAT TATCAGACAA 900 CTCAAGGATA ATCATGAGGT CTACAGTCCA AGGACACGGA ACGCGATCAT AAGTGACGCA 960 TTTGCGGCAG CACTGCTTGA CAATGGGCTC AAGTATGAGA CTGTTTTCGA ACTATTGGAA 1020 TACGCAAAGA ACGAACAGGA ATATCTTCCA TGGGATGAAA TCATTTCTGG ATTCTATTCA 1080 ATTCTTGAAT TCTTTGGCAA CGAGCCAGAG TCAAAATGGG CTAAAAGCTA TATGATGAAC 1140 ATATTGAAGC CGATGTATGA CAATAGCAGT ATGCAGTACA TCGCTGACAA CTACAAAAAC 1200 GATTCCCTAT TTTTCGAAAA CAATCTCCAA AAAGCGGTCA TTGATGCGTA CTGTCGCCTT 1260 GGATCAAAAG ATTGCATAGG AAAGTATAAG GATCTCTTCA TTAAAGAAGT CATGGAAAAA 1320 TGCGAGGATG ATGAAGAGGC AAGCAAATGC GTGACGGTTG CCGCTCCTCT GCGATCGAG3 1380 GTTTACTGCT ATGGTGTGAA AGAGGGCGGA GACGATGCTT TCGACAAGGT TATGGGGCTG 1440 TACAGTGCAG AAAATGTTCA GCTGGAGAAG GACATTCTGC TTCGAGCGCT AGGATGTCAC 1500 CGAGATATCA CAGCTCTAAA AGGGTTACTT CTGCGAGCGC TGGATCGGAA TTCGTCGTTT 1560 GTTCGCCTTC AAGATGTGTC TGACGTCTTT ATGGCTGTAT CTGGAAAGCC CGTGGGCGAG 1620 GAATTCATGT TCAACTTCCT TCTAGAGAGA TGGGAAGAAA TAGTCGAAAG CTTACCGTCG 1680 GAACACACTT CAGTTGAAAA AGTGATCAGG GACTGTTCTA CAGGCATTCG ATCCGAGCAA 1740 CAAATAGAAC AGTTGAGAAA TCTTCACAAA AATGGCCGAA ATGCTCGAGA TTACGGTGCA 1800 TTCGACGAGG GAATCGAACG AGCAGAGCAC AAAGTCGACT GGATAAAA 1848 (2) INFORMATION FOR SEQ ID NO: 22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1584 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: GGAGCGATGG AGAACTGGGG GCTTATCACT TACAGAGAAA CTGCTCTGTT GTACGATGAA 60 AGACTCTACG CACCGGTTAA TAAGAAAAGA GTTGCTTCAG TGGTTGCTCA CGAACTTTCT 120 CA.TCAATGGT TCGGCGATCT TGTTACAATG GAATGGTGGG ATAATCTGTG GTTGAATGAA 180 GGTTTTGCAT CGTTCATGCA ATATATTGGC ACAAATGAGA TTACCCGCGA TAACTTTAAG 240 ATGAAGGACT ATTTCCTGGT GGACTCGTTT GCGCAAGGCA TGGAAGCTGA CGTAGCTTCA 300 AGCCATCCGC TATCCTTTAA AGTCGACAAG GCTGCAGATG TTGTTGAAGC TTTTGATGAT 360 GTCACTTATC GTAAAGGAGC ATCGATTCTC ACAATGCTAC AAGCGTTAAT TGGTGAACAA 420 AACTTCCAAC GGGCCATAAG GCAATACCTC ATAAAGTTCT CGTACAATAA TGCGAACGCT 480 TCCGATCTCT GGAATGTTTT TGACGAAGTT GTCAAGGATG TTGCGGGACC TGACGGTAGC 540 CGCATGAAAA CCTCTGAATT TGCCGATCAG TGGACGACTC AGATGGGCTA CCCAGTAGTT 600 ACAGTGGAAA CGTTCAATGC AACTACCTTC AAAATATCAC AAAGTCGATA CAAGAAGAAC 660 AAGAACGCTC AGGAGCCGGA AAAATATCGT CATCCAAAAT ATGGGTTTGA ATGGGATGTT 720 CCTGTATGGT ACCAGGACAG CAAAAACAGC GATGTGAAGC GGATCTGGTT AACCAGGGAC 780 AAGCCGACTT ATTTGCATGT GAACAGTTCC GATGCGTCCA TCGTTGTGAA CGCCGACAGG 840 CATGGATTTT ACCGGCAAAA CTACGATGAA GATGGCTGGC GAAAGATTAT AAAGATACTC 900 AAGGATAATC ATAAGTACTA TAGTCCGAGA ACCAGGAACG CTATCATAAG CGATGCGTTT 960 GCAGCTGCCC TTATTGACAA ACTTGAGTAC GAGACTGTTT TTGATCTTCT GGAATACGCT 1020 AATCAAGAAG AGGAATTTCT ACCGTGGAAC GAGATTATAA CTGGATTCTA TTCAATTTTG 1080 GAGTACTTTG GCAGCGAGCC GGAATCAAAG TTCGCAAAGA ACTACATGAT GAGCATTCTG 1140 .AAACCAATGT ACGACAAGAG CAGCGTTGAT TATATCGCCG AGAACTACAC GAACGATTCG 1200 CTATTTTTCG AAAACAATCT TCAAAAGGCA ATTATCGAGG CCTACTGCTA TTTTGGATCG 1260 AAGGGCTGTA TCCAAAAGTT TAAAGAACTC TTCGACAAAG AGGTTGTGCA GAAATGCAAG 1320 GGCGGTCACA AGGCAAGCAA ATGTGTGGAC GTTGCTGCTC CTCTTCGAGC GATGGTTTAC 1380 TGCTATGGTG TGAACGAAGG CGGAGATGAT GCATACGACA AGGTGATGGA GTTATATTAC 1440 GCAGAAACTG TTCAGTTGGA GAAGGACTAC CTTCTCGGAG CTTTAGCATG TCACAAAGAC 1500 ATCACAGCCT TGAAAGGACT TCTTCTGCTG GCTCTGGATC GTAATTCGTC GTTTGTTCGC 1560 CTTCAAGATA TGGCCAACGT TTCT 1584 (2) INFORMATION FOR SEQ ID NO: 23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1848 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: GGTGCGATGG AGAACTGGGG GCTCATCACT TACAGAGAAA ATTCCTTGCT GTACGATGAA 60 AGATACTATG CACCGTTGAG CAAGGAACGG GTTGCCATTG TGGTTGCTCA TGAACTCGCC 120 CATCAGTGGT TCGGCAACCT TGTCACGTTG AAGTGGTGGG ATGATTTGTG GCTAAACGAA 180 GGTTTCGCAA .CATTTGTTGA ATTCATCGGT GCAGATCATA TCAGCAACGG AACTTTCAGA 240 ATGAAGGACT ATTTTCTATT GGATGCTCTT GTAGACGCTT TGGAGGCGGA TTCGGTAGCT 300 TCAAGCCATC CGCTATCATT TAAAATCGAT AAAGCTTCGG AGGTTTACGA AGCTTTCGAC 360 GCTATCACGT ACTCAAAAGG AGCGTCAGTT CTCACAATGC TGCAAGCTTT GATTGGTGAA 420 GAAAACTTCA AGCGGGCTGT GACGCAATAT CTCAATAAGT TTTCGTTCGA TAACGCGAAA 480 GCTTCCGATC TTTGGGGTGT CTTCGATGAA GTTGTAAAGG ATGTCAAGGG CCCCGACGGT 540 AATCCTATGA AAATCACTGA ATTCGCTTAT CAGTGGACGA CTCAGATGGG CTACCCAATA 600 GTCACAGTGG AAACGTTCAA CGCAACTTCT TTGAAAGTCT CACAAAACCG ATACAAGACC 660 AATAAGGACG CCCAAGAGCC GGAAAAGTAC CGTCATCCGA AATATGGGTT CAAGTGGGAT 720 GTTCCTCTAT GGTATCAAGA AGGCGAAAGC AAGGAAATAA AGCAGACCTG GTTGACTAGA 780 GGAGAGCCCC TTTATTTGCA CGTGAGCAGT TCGGATGATT CCATTGTGGT GAACGCCCAT 840 CGGCATGGAT TTTACAGGCA AAACTACGAT GCCAACGGTT GGCGAAAGAT TATCAGGCAA 900 CTCAAGGAAA ATCATGAGGT CTACAGTCCA AGGACACGGA ACGCGATCAT AAGTGACGCA 960 TTTGCGGCAG CACTGCTTGA CAATGGGCTC GAGTATGAGA CTGTTTTCGA ACTATTGGAA 1020 TACGCAAAGA ACGAACAGGA ATATCTTCCA TGGGATGAAA TCATTTCTGG ATTCTATTCA 1080 ATTCTTGAAT TCTTTGGCAA CGAGCCAGAG TCAAAATGGG CCAAAGCGTA TACGATGAGC 1140 ATATTGAAGC CGATGTATGA CAATAGCAGT ATGCAGTACA TCGCTGAAAA CTACAAGAAC 1200 GATTCTCTAT TTTTTGAAAA CAATCTCCAG AAAGCAGTCA TTGATGCGTA CTGTCGCCTT 1260 GGATCAAAAG ATTGCATAGG AAAGTATAAG GATCTCTTCG TCAAAGAAGT CATGGAAAAA 1320 TGCGAAGATG GTGAAGAGGC AAGCAAATGC GTGACGGTTG CCGCTCCTCT TCGATCGAGA 1380 GTTTACTGCT ATGGTGTGAA AGAGGGCGGA GACGATGCTT TCGACAAGGT TATGGGGCTT 1440 TACTCTGCAG AAAATGTTCA GCTGGAGAAG GACATTCTGC TTCGAGCGCT AGGATGTCAC 1500 CGAGATATCA CAGCTCTAAA AGGATTACTT CTTCGAGCGC TGGATCGGAA TTCGTCGTTT 1560 GTTCGCCTTC AAGATGTGTC TGACGTCTTT ATGGCTGTAT CAGGAAATCC CGTGGGCGAG 1620 GAATTCATGT TCAACTTCCT TCTAGAGAGA TGGGAAGAAA TAATCGAAAG CTTACCGTCG 1680 GAACACACTT CAGTTGAAAA AGTGATCAAG GACTGTTCTA CAGGCATTCG ATCCGAGCAA 1740 CAAATAGAAC AGTTGAGAAA TCTTCACAAA AATGGCCGAA ATGCTCGAGA TTACGGTGCA 1800 TTTGACGAGG GAATCGAACG AGCAGAGCAC AAAGTCGACT GGTACAAA 1848 (2) INFORMATION FOR SEQ ID NO: 24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 346 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: Met Thr Ala Gin Trp Ala Lys Arg Thr Val Leu Arg Phe Thr Pro lie 1 5 10 15 Ser Leu Leu Val Ala Ser Leu Ala Met Ala Leu Ala lie Ala Leu Ser 20 25 30 lie Gly Leu Thr Tyr His Phe Val Arg Asn Ala Tyr Asp Thr Ser His 35 40 45 Asn Gly Lys Asp His Pro Gly Gly Asp Asp Asn Ser Pro Ser Ala Glu 50 55 60 Glu Leu Arg Leu Pro Arg Ser Val lie Pro Leu Leu Tyr Asp Leu Ser 65 70 75 80 lie Lys Thr Tyr Leu Pro Asn Tyr Val Asp Phe Pro Pro Glu Lys Asn 85 90 95 Leu Thr Phe Asp Gly Gin Val Glu lie Ser Met Val Val Met Glu Pro 100 105 110 Thr Arg Ser lie Val Leu Asn Ala Lys Asn lie Thr Val lie Pro Glu 115 120 125 Lys Cys Glu Val Phe Ser Gly Asp Glu Lys Leu Glu lie Glu Ser Val 130 135 140 Met Glu His Glu Arg Leu Glu Lys Leu Glu Phe Leu Leu Lys Lys Arg 145 150 155 160 Leu Glu Lys Asp Gin Lys Val Leu Leu Lys Val lie Tyr lie Gly Leu 165 170 175 lie Ser Asn Thr Leu Gly Gly Leu Tyr Gin Ala Thr Tyr Thr His Thr 180 185 190 Asp Gly Thr Thr Lys lie Ala Ala Ala Ser Gin Met Glu Pro Thr Asp 195 200 205 Ala Arg Arg Met Val Pro Cys Leu Asp Glu Pro Asn Tyr Lys Ala Asn 210 215 220 Trp Thr Val Thr Val lie His Pro Lys Gly Thr Arg Ala Val Ser Asn 225 230 235 240 Gly lie Glu Thr Asn Gly Glu Gly Asp lie Asn Gly Asp Trp lie lie 245 250 255 Ser Lys Phe Glu Thr Thr Pro Arg Met Ser Ser Tyr Leu Leu Ala Val 260 265 270 lie Val Ser Glu Phe Asp Phe lie Glu Gly Asn Thr Thr Ser Gly Val 275 280 285 Arg Phe Arg lie Trp Ser Arg Pro Glu Ala Lys Asn Met Thr Gin Tyr 290 295 300 Ala Leu Asp Ala Gly lie Arg Cys Leu Glu Phe Tyr Glu Ser Phe Phe 305 310 315 320 Gly lie Lys Phe Pro Leu Gin Lys Gin Asp Met Val Ala Leu Pro Asp 325 330 335 Phe Ser Ala Gly Ala Met Glu Asn Trp Gly 340 345 (2) INFORMATION FOR SEQ ID NO: 25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 350 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25: Met Glu Glu Pro Arg Ala Arg Arg Arg Thr Val Leu Arg Leu Thr Pro 1 5 10 15 lie Thr Leu Ser lie Ala Leu Leu Gly lie Ala Val Ala Val Ala Leu 20 25 30 Ser lie Gly Leu Thr Tyr Leu Phe Thr Arg Asn Ala Tyr Asp Thr Ser 35 40 45 Arg Lys Pro Lys Glu Pro Asp His Pro Gly Gly Gly Asp Asp Asn Pro 50 55 60 Pro Ser Ala Glu Glu Leu Arg Leu Pro Thr Asn lie Lys Pro Leu Leu 65 70 75 - 80 Tyr Asn Leu Thr lie Lys Thr Tyr Leu Pro Gly.Tyr Val Asp Phe Pro 85 90 95 Ser Glu Lys Asn Leu Thr Phe Asp Ala Gin Val Leu lie Ser Met Val 100 105 110 Val Val Glu Pro Thr Lys Ser lie Val Leu Asn Ala Lys Lys lie Thr 115 120 125 Ala Leu Pro Lys Glu Cys Glu Val Phe Ala Gly Asp Gin Lys Leu Asp 130 135 140 lie Glu Ser Val Thr Asp His Glu Arg Leu Glu Lys Leu Glu Phe Thr 145 150 155 160 Leu Lys Gin Gin Leu Glu Lys Asp Gin Lys lie Gin Leu Lys Val Val 165 170 175 Tyr Ser Gly Leu lie Ser Asp Thr Leu Gly Gly Leu Tyr Gin Ala Thr 180 185 190 Tyr Lys Asp Thr Asp Gly Thr Thr Lys He Val Ala Val Ser Gin Met 195 200 205 Glu Pro Thr Asp Ala Arg Arg Met Val Pro Cys Phe Asp Glu Pro Ser 210 215 220 Phe Lys Ala Asn Trp Thr Val Thr He He His Pro Arg Gly Thr Thr 225 230 235 240 Ala Ala Ser Asn Gly He Glu Thr Asn Gly Glu Gly Glu Pro Asp Gly 245 250 255 Asp Trp He Thr Ser Lys Phe Lys Thr Thr Pro Arg Met Ser Ser Tyr 260 265 270 Leu Leu Ala Val He Val Ser Glu Phe Lys Tyr He Glu Gly Arg Thr 275 280 285 Lys Ser Gly Val Arg Phe Arg He Trp Ser Arg Pro Glu Ala Val Lys 290 295 300 Met Thr Lys Phe Ala Leu Asp Ala Gly Val Arg Cys Leu Glu Phe Tyr 305 310 315 320 Glu Lys Phe Phe Asp He Arg Phe Pro Leu Glu Lys Gin Asp Met Val 325 330 335 Ala Leu Pro Asp Phe Ser Ala Gly Ala Met Glu Asn Trp Gly 340 345 350 (2) INFORMATION FOR SEQ ID NO: 26: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 616 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: Gly Ala Met Glu Asn Trp Gly Leu He Thr Tyr Arg Glu Asn Ser Leu 1 5 10 15 Leu Tyr Asp Glu Arg Tyr Tyr Ala Pro Leu Asn Lys Glu Arg Val Ala 20 25 - 30 He Val Val Ala His Glu Leu Ala His Gin Trp.Phe Gly Asn Leu Val 35 40 45 Thr Leu Lys Trp Trp Asp Asp Leu Trp Leu Asn Glu Gly Phe Ala Thr 50 55 60 Phe Val Glu Phe He Gly Ala Asp His He Ser Asn Gly Thr Phe Arg 65 70 75 80 Met Lys Asp Tyr Phe Arg Leu Asp Ala Leu Val Asp Ala Leu Glu Ala 85 90 95 Asp Ala Val Ala Ser Ser His Pro Leu Ser Phe Lys He Asp Lys Ala 100 105 110 Ser Glu Val Tyr Glu Ala Phe Asp Ala He Thr Tyr Ser Lys Gly Ala 115 120 125 Ser Val Leu Thr Met Leu Gin Ala Leu He Gly Glu Glu Asn Phe Lys 130 135 140 Gin Ala Val Thr Gin Tyr Leu Asn Lys Phe Ser Phe Asp Asn Ala Lys 145 150 155 160 Ala Ser Asp Leu Trp Gly Val Phe Asp Glu Val Val Lys Asp Val Lys 165 170 175 Gly Pro Asp Gly Asn Pro Met Lys Thr Thr Glu Phe Ala Tyr Gin Trp 180 185 190 Thr Thr Gin Met Gly Tyr Pro He Val Thr Val Glu Thr Phe Asn Ala 195 200 205 Thr Ser Leu Lys Val Ser Gin Asn Arg Tyr Lys Thr Asn Lys Asp Ala 210 215 220 Gin Glu Pro Glu Lys Tyr Arg His Pro Lys Tyr Gly Phe Lys Trp Asp 225 230 235 240 Val Pro Leu Trp Tyr Gin Glu Gly Glu Ser Asn Glu He Lys Gin Thr 245 250 255 Trp Leu Thr Arg Gly Glu Pro Leu Tyr Leu His Val Ser Ser Ser Asp 260 265 270 Asp Ser He Val Val Asn Ala Asp Arg His Gly Phe Tyr Arg Gin Asn 275 280 285 Tyr Asp Ala Asn Gly Trp Arg Lys He He Arg Gin Leu Lys Asp Asn 290 295 300 His Glu Val Tyr Ser Pro Arg Thr Arg Asn Ala He He Ser Asp Ala 305 310 315 320 Phe Ala Ala Ala Leu Leu Asp Asn Gly Leu Lys Tyr Glu Thr Val Phe 325 330 335 Glu Leu Leu Glu Tyr Ala Lys Asn Glu Gin Glu Tyr Leu Pro Trp Asp 340 345 350 Glu He He Ser Gly Phe Tyr Ser He Leu Glu Phe Phe Gly Asn Glu 355 360 365 Pro Glu Ser Lys Trp Ala Lys Ser Tyr Met Met Asn He Leu Lys Pro 370 375 380 Met Tyr Asp Asn Ser Ser Met Gin Tyr He Ala Asp Asn Tyr Lys Asn 385 390 395 400 Asp Ser Leu Phe Phe Glu Asn Asn Leu Gin Lys Ala ' Val He Asp Ala 405 410 415 Tyr Cys Arg Leu Gly Ser Lys Asp Cys He Gly Lys Tyr Lys Asp Leu 420 425 430 Phe He Lys Glu Val Met Glu Lys Cys Glu Asp Asp Glu Glu Ala Ser 435 440 445 Lys Cys Val Thr Val Ala Ala Pro Leu Arg Ser Arg Val Tyr Cys Tyr 450 455 460 Gly Val Lys Glu Gly Gly Asp Asp Ala Phe Asp Lys Val Met Gly Leu 465 470 475 480 Tyr Ser Ala Glu Asn Val Gin Leu Glu Lys Asp He Leu Leu Arg Ala 485 490 495 Leu Gly Cys His Arg Asp He Thr Ala Leu Lys Gly Leu Leu Leu Arg 500 505 510 Ala Leu Asp Arg Asn Ser Ser Phe Val Arg Leu Gin Asp Val Ser Asp 515 520 525 Val Phe Met Ala Val Ser Gly Lys Pro Val Gly Glu Glu Phe Met Phe 530 535 540 Asn Phe Leu Leu Glu Arg Trp Glu Glu He Val Glu Ser Leu Pro Ser 545 550 555 560 Glu His Thr Ser Val Glu Lys Val He Arg Asp Cys Ser Thr Gly He 565 570 575 Arg Ser Glu Gin Gin He Glu Gin Leu Arg Asn Leu His Lys Asn Gly 580 585 590 Arg Asn Ala Arg Asp Tyr Gly Ala Phe Asp Glu Gly He Glu Arg Ala 595 600 605 Glu His Lys Val Asp Trp He Lys 610 615 (2) INFORMATION FOR SEQ ID NO: 27: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 528 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: Gly Ala Met Glu Asn Trp Gly Leu He Thr Tyr Arg Glu Thr Ala Leu 10 15 Leu Tyr Asp Glu Arg Leu Tyr Ala Pro Val Asn Lys Lys Arg Val Ala 20 25 30 Ser Val Val Ala His Glu Leu Ser His Gin Trp Phe Gly Asp Leu Val 35 40 45 Thr Met Glu Trp Trp Asp Asn Leu Trp Leu Asn Glu Gly Phe Ala Ser 50 55 60 Phe Met Gin Tyr He Gly Thr Asn Glu He Thr Arg Asp Asn Phe Lys 65 70 75 80 Met Lys Asp Tyr Phe Leu Val Asp Ser Phe Ala Gin Gly Met Glu Ala 85 90 - 95 Asp Val Ala Ser Ser His Pro Leu Ser Phe Lys.Val Asp Lys Ala Ala 100 105 110 Asp Val Val Glu Ala Phe Asp Asp Val Thr Tyr Arg Lys Gly Ala Ser 115 120 125 He Leu Thr Met Leu Gin Ala Leu He Gly Glu Gin Asn Phe Gin Arg 130 135 140 Ala He Arg Gin Tyr Leu He Lys Phe Ser Tyr Asn Asn Ala Asn Ala 145 150 155 160 Ser Asp Leu Trp Asn Val Phe Asp Glu Val Val Lys Asp Val Ala Gly 165 170 175 Pro Asp Gly Ser Arg Met Lys Thr Ser Glu Phe Ala Asp Gin Trp Thr 180 185 190 Thr Gin Met Gly Tyr Pro Val Val Thr Val Glu Thr Phe Asn Ala Thr 195 200 205 Thr Phe Lys He Ser Gin Ser Arg Tyr Lys Lys Asn Lys Asn Ala Gin 210 215 220 Glu Pro Glu Lys Tyr Arg His Pro Lys Tyr Gly Phe Glu Trp Asp Val 225 230 235 240 Pro Val Trp Tyr Gin Asp Ser Lys Asn Ser Asp Val Lys Arg He Trp 245 250 255 Leu Thr Arg Asp Lys Pro Thr Tyr Leu His Val Asn Ser Ser Asp Ala 260 265 270 Ser He Val Val Asn Ala Asp Arg His Gly Phe Tyr Arg Gin Asn Tyr 275 280 285 Asp Glu Asp Gly Trp Arg Lys He He Lys He Leu Lys Asp Asn His 290 295 300 Lys Tyr Tyr Ser Pro Arg Thr Arg Asn Ala He He Ser Asp Ala Phe 305 310 315 320 Ala Ala Ala Leu He Asp Lys Leu Glu Tyr Glu Thr Val Phe Asp Leu 325 330 335 Leu Glu Tyr Ala Asn Gin Glu Glu Glu Phe Leu Pro Trp Asn Glu He 340 345 350 He Thr Gly Phe Tyr Ser He Leu Glu Tyr Phe Gly Ser Glu Pro Glu 355 360 365 Ser Lys Phe Ala Lys Asn Tyr Met Met Ser He Leu Lys Pro Met Tyr 370 375 380 Asp Lys Ser Ser Val Asp Tyr He Ala Glu Asn Tyr Thr Asn Asp Ser 385 390 395 400 Leu Phe Phe Glu Asn Asn Leu Gin Lys Ala He He Glu Ala Tyr Cys 405 410 415 Tyr Phe Gly Ser Lys Gly Cys He Gin Lys Phe Lys Glu Leu Phe Asp 420 425 430 Lys Glu Val Val Gin Lys Cys Lys Gly Gly His Lys Ala Ser Lys Cys 435 440 445 Val Asp Val Ala Ala Pro Leu Arg Ala Met Val Tyr Cys Tyr Gly Val 450 455 460 Asn Glu Gly Gly Asp Asp Ala Tyr Asp Lys Val Met Glu Leu Tyr Tyr 465 470 475 480 Ala Glu Thr Val Gin Leu Glu Lys Asp Tyr Leu Leu Gly Ala Leu Ala 485 490 495 Cys His Lys Asp He Thr Ala Leu Lys Gly Leu Leu Leu Leu Ala Leu 500 505 510 Asp Arg Asn Ser Ser Phe Val Arg Leu Gin Asp Met Ala Asn Val Ser 515 520 525 (2) INFORMATION FOR SEQ ID NO: 28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 616 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: Gly Ala Met Glu Asn Trp Gly Leu He Thr Tyr Arg Glu Asn Ser Leu 1 5 10 15 Leu Tyr Asp Glu Arg Tyr Tyr Ala Pro Leu Ser Lys Glu Arg Val Ala 20 25 30 He Val Val Ala His Glu Leu Ala His Gin Trp Phe Gly Asn Leu Val 35 40 45 Thr Leu Lys Trp Trp Asp Asp Leu Trp Leu Asn Glu Gly Phe Ala Thr 50 55 60 Phe Val Glu Phe He Gly Ala Asp His He Ser Asn Gly Thr Phe Arg 65 70 75 80 Met Lys Asp Tyr Phe Leu Leu Asp Ala Leu Val Asp Ala Leu Glu Ala 85 90 95 Asp Ser Val Ala Ser Ser His Pro Leu Ser Phe Lys He Asp Lys Ala 100 105 110 Ser Glu Val Tyr Glu Ala Phe Asp Ala He Thr Tyr Ser Lys Gly Ala 115 120 125 Ser Val Leu Thr Met Leu Gin Ala Leu He Gly Glu Glu Asn Phe Lys 130 135 140 Arg Ala Val Thr Gin Tyr Leu Asn Lys Phe Ser Phe Asp Asn Ala Lys 145 150 155 160 Ala Ser Asp Leu Trp Gly Val Phe Asp Glu Val Val Lys Asp Val Lys 165 170 175 Gly Pro Asp Gly Asn Pro Met Lys He Thr Glu Phe Ala Tyr Gin Trp 180 185 190 Thr Thr Gin Met Gly Tyr Pro He Val Thr Val Glu Thr Phe Asn Ala 195 200 205 Thr Ser Leu Lys Val Ser Gin Asn Arg Tyr Lys Thr Asn Lys Asp Ala 210 215 220 Gin Glu Pro Glu Lys Tyr Arg His Pro Lys Tyr Gly Phe Lys Trp Asp 225 230 235 240 Val Pro Leu Trp Tyr Gin Glu Gly Glu Ser Lys Glu He Lys Gin Thr 245 250 255 Trp Leu Thr Arg Gly Glu Pro Leu Tyr Leu His Val Ser Ser Ser Asp 260 265 270 Asp Ser He Val Val Asn Ala His Arg His Gly Phe Tyr Arg Gin Asn 275 280 285 Tyr Asp Ala Asn Gly Trp Arg Lys He He Arg Gin Leu Lys Glu Asn 290 295 300 His Glu Val Tyr Ser Pro Arg Thr Arg Asn Ala He He Ser Asp Ala 305 310 315 320 Phe Ala Ala Ala Leu Leu Asp Asn Gly Leu Glu Tyr Glu Thr Val Phe 325 330 335 Glu Leu Leu Glu Tyr Ala Lys Asn Glu Gin Glu Tyr Leu Pro Trp Asp 340 345 350 Glu He He Ser Gly Phe Tyr Ser He Leu Glu Phe Phe Gly Asn Glu 355 360 365 Pro Glu Ser Lys Trp Ala Lys Ala Tyr Thr Met Ser He Leu Lys Pro 370 375 380 Met Tyr Asp Asn Ser Ser Met Gin Tyr He Ala Glu Asn Tyr Lys Asn 385 390 395 400 Asp Ser Leu Phe Phe Glu Asn Asn Leu Gin Lys Ala Val He Asp Ala 405 410 415 Tyr Cys Arg Leu Gly Ser Lys Asp Cys He Gly Lys Tyr Lys Asp Leu 420 425 430 Phe Val Lys Glu Val Met Glu Lys Cys Glu Asp Gly Glu Glu Ala Ser 435 440 445 Lys Cys Val Thr Val Ala Ala Pro Leu Arg Ser Arg Val Tyr Cys Tyr 450 455 460 Gly Val Lys Glu Gly Gly Asp Asp Ala Phe Asp Lys Val Met Gly Leu 465 470 475 480 Tyr Ser Ala Glu Asn Val Gin Leu Glu Lys Asp He Leu Leu Arg Ala 485 490 495 Leu Gly Cys His Arg Asp He Thr Ala Leu Lys Gly Leu Leu Leu Arg 500 505 510 Ala Leu Asp Arg Asn Ser Ser Phe Val Arg Leu Gin Asp Val Ser Asp 515 520 525 Val Phe Met Ala Val Ser Gly Asn Pro Val Gly Glu Glu Phe Met Phe 530 535 540 Asn Phe Leu Leu Glu Arg Trp Glu Glu He He Glu Ser Leu Pro Ser 545 550 555 560 Glu His Thr Ser Val Glu Lys Val He Lys Asp Cys Ser Thr Gly He 565 570 575 Arg Ser Glu Gin Gin He Glu Gin Leu Arg Asn Leu His Lys Asn Gly 580 585 590 Arg Asn Ala Arg Asp Tyr Gly Ala Phe Asp Glu Gly He Glu Arg Ala 595 600 605 Glu His Lys Val Asp Trp Tyr Lys 610 615

Claims: Claims : 1. A protective helminth parasite antigen which is characterised by possesεing aminopeptidase-like activity and which, in native form, is an integral membrane protein associated with the gut microvilli of a non- obligate blood feeding helminth parasite, or a functionally-equivalent variant, antigenic fragment or precursor thereof. 2. A method of preparing an antigen, or a functionally- equivalent variant, antigenic fragment or precursor thereof as claimed in claim l, comprising the step of subjecting a crude extract of a non-obligate blood feeding helminth parasite to extraction with a detergent. 3. A method of preparing an antigen, or a functionally- equivalent variant, antigenic fragment or precursor thereof as claimed in claim 2, comprising the steps of subjecting a crude extract of a non-obligate blood feeding helminth parasite to extraction with a first detergent to remove membrane-associated proteins followed by solubilization with a second detergent to recover integral membrane proteins. 4. A method as defined in claim 3 wherein the first detergent is Tween 20 and the second detergent is Thesit. 5. A method for preparing truncated form of an antigen, or a functionally-equivalent variant or precursor thereof, as claimed in claim 1, said method comprising releasing the truncated form of the antigen from membranes by treatment with a proteolytic enzyme. 6. A method of preparing an antigen, or a functionally- equivalent variant, antigenic fragment or precursor thereof, as claimed in any one of claims 2 to 5 wherein the method additionally compriseε at leaεt one additional chromatographic purification step. 7. A method of preparing an antigen, or a functionally- equivalent variant, antigenic fragment or precursor thereof, as claimed in claim 1, said process comprising preparing an extract of said parasite containing at least one protective antigen as defined in claim 1, purifying said antigen therefrom by binding said antigen to an immobilized phase including a specific binding partner for the antigen and subsequently eluting said antigen from said immobilized phase. 8. A method of preparing an antigen, or a functionally- equivalent variant, antigenic fragment or precursor thereof, as claimed in claim 1, wherein said method comprises chemical synthesis. 9. An antigen, or a functionally-equivalent variant, antigenic fragment or precursor thereof, prepared by any one of the methods defined in claims 2 to 8. 10. An antigen, or a functionally-equivalent variant, antigenic fragment or precursor thereof, as defined in claim l or 9 wherein the helminths are nematodes. 11. An antigen, or a functionally-equivalent variant, antigenic fragment or precursor thereof, as defined in claim 10 wherein the nematodes are Ostertaσia or Cooperia• 12. An antigen, or a functionally-equivalent variant, antigenic fragment or precursor thereof, as defined in claim 11 wherein said antigen is obtainable from Ostertagia and has an apparent molecular weight (Mr) of about 124,000 to 126,000 as determined by SDS-PAGE on a 6-16% gel under denaturing conditionε. 13. An antigen, or a functionally-equivalent variant, antigenic fragment or precursor thereof, as defined in claim 1 or 9 wherein the amino acid sequence of the antigen contains the sequence: A E D L R L P T N I R P L I Y D L T (Sequence I.D. No. 1). 14. An antigen, or a functionally-equivalent variant, antigenic fragment or precursor thereof, as claimed in any one of claims 1 or 9 to 13, for use in stimulating an immune response against helminth parasites in a human or non-human animal. 15. The use of a helminth parasite antigen, or a functionally-equivalent variant, antigenic fragment or precursor thereof, as claimed in any one of claims 1 or 9 to 13, for the preparation of vaccine compositions for use in stimulating an immune response against helminth parasites in a human or non-human, animal. 16. A vaccine composition for stimulating an immune response against helminth parasites in a human or non- human animal comprising one or more antigens, antigenic fragments, precursors or functionally-equivalent variants thereof as claimed in any one of claims 1 or 9 to 13, together with a pharmaceutically acceptable carrier or diluent. 17. A method of stimulating an immune response against helminth parasites in a human or non-human animal, comprising adminiεtering to said animal a vaccine composition as claimed in claim 16. 18. An antibody or the antigen-binding fragment thereof specifically binding to an antigen, or a functionally- equivalent variant, antigenic fragment or precursor thereof as claimed in any one of claims 1 or 9 to 13, or to the idiotype of a said antigen-binding antibody. 19. A vaccine composition containing an antibody or fragment thereof as claimed in claim 18. 20. A nucleic acid molecule comprising a nucleotide sequence encoding an antigen, or a functionally- equivalent variant, antigenic fragment or precursor thereof as claimed in any one of claims 1 or 9 to 13. 21. A nucleic acid molecule as claimed in claim 20 which comprises one or more nucleotide sequences substantially corresponding to all or a portion of a nucleotide sequence shown in Figure 19 and/or 20 (Sequence I.D. Nos. 19-23) or a sequence which is degenerate or substantially homologous with or which hybridises with said sequence. 22. A cloning or expression vector containing a nucleic acid molecule as claimed in claim 20 or 21. 23. A host cell or organism transformed with or containing a nucleic acid molecule as claimed in claim 20 or 21. 24. A method of preparing an antigen, or a functionally-equivalent variant, antigenic fragment or precursor thereof, as claimed in any one of claims 1 or 9 to 13, wherein said method comprises culturing a host cell containing a nucleic acid molecule encoding all or a portion of said antigen, under conditions whereby said antigen is expressed and recovering said antigen thus produced. 25. A synthetic polypeptide or fragment thereof expresεed by a hoεt cell or organiεm as claimed in claim 23.

Inventors: GRAHAM MARGARET, MCMICHAEL-PHILLIPS DANIELLE, MUNN EDWARD ALBERT, GRAHAM, MARGARET, MCMICHAEL-PHILLIPS, DANIELLE, MUNN, EDWARD, ALBERT
Applicants: BIOTECH & BIOLOG SCIEN RES, GRAHAM MARGARET, MCMICHAEL PHILLIPS DANIELLE, MUNN EDWARD ALBERT, BIOTECHNOLOGY AND BIOLOGICAL SCIENCES RESEARCH COU, GRAHAM, MARGARET, MCMICHAEL-PHILLIPS, DANIELLE, MUNN, EDWARD, ALBERT
Application year: 1994
Main classification: C12N15
IPC codes: A61K39, C07K14, C12N9, C12N15